Methods of treating or preventing pyruvate kinase deficiency

ABSTRACT

The present disclosure provides polynucleotide cassettes, expression vectors and methods for the expression of a gene in mammalian cells to provide gene therapy for pyruvate kinase deficiency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/US2017/028695, filed on Apr. 20, 2017, which claims priority to U.S.Provisional Application No. 62/325,397, filed on Apr. 20, 2016, which isincorporated by reference herein in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ROPA_001_01US_ST25.txt. The text file is 6,288bytes, created on Apr. 2, 2019, and is submitted electronically viaEFS-Web.

FIELD OF THE INVENTION

This invention pertains to gene therapy of Pyruvate Kinase Deficiency.

BACKGROUND OF THE INVENTION

Pyruvate Kinase Deficiency (PKD) is a monogenic metabolic disease causedby mutations in the PKLR gene that leads to hemolytic anemia of variablesymptomatology and that can be fatal during the neonatal period. PKDrecessive inheritance trait and its curative treatment by allogeneicbone marrow transplantation provide an ideal scenario for developinggene therapy approaches.

Among many other hereditary enzymatic defects affecting theerythrocytes, Pyruvate Kinase deficiency (PKD) is the most frequent onecausing chronic nonspherocytic hemolytic anemia (CNSHA) (Zanella et al.2007). Onset and severity of PKD are very variable and range from mildto severe neonatal anemia, becoming fatal during the childhood in themost severe cases (Pissard et al 2006). Growth retardation, Hydropsfetalis and death during the neonatal period have also been reportedwith low frequency (Gilsanz et al. 1993). PKD prevalence has beenestimated at 1:20,000 in the general Caucasian population (Beutler et al2000) and, so far, more than 195 different mutations in the PKLR genehave been identified (lovd.nl/pklr). Allogeneic bone marrowtransplantation (BMT) has been successfully used to cure severe PKDpatients (Tanphaichitr et al 2000), but the low availability ofhistocompatible donors and the serious complications associated with theBMT of these patients (i.e. graft versus host disease, opportunisticinfections, etc.) make periodic blood transfusions and splenectomy themain therapeutic options for most of the severe forms of PKD (Zanella etal 2005), dramatically increasing patient morbidity and mortality(Hilgard et al 2005). The limited efficacy and side effects of thetherapeutic options for severe PKD patients and its recessiveinheritance trait make PKD a suitable disease to be treated by genetherapy.

PKD is caused by defects in the Pyruvate Kinase (PK) enzyme (Zanella2005) that catalyses the last ATP-generating reaction of the glycolysispathway in all cells. In mature erythrocytes PK becomes essential asRBCs only express the R-type specific isoform (RPK) (Kanno et al 1992)due to the regulation of the erythroid specific alternative promoter ofthe PKLR locus (Noguchi et al 1987). Thus, any loss of RPK activityimpairs RBC metabolism and lifespan (Zanella 2005), leading to CNSHA.

A promising approach to treating and preventing genetic and otherdiseases and disorders is delivery of therapeutic agents with a genetherapy vector. Currently, viral vectors show the greatest efficiency ingene transfer, and for correction of genetic diseases such thatpersistent gene expression is required, herpesvirus, retrovirus,lentivirus, adenovirus, or AAV based vectors are desirable due to theintegrating nature of the viral life cycle.

Gene therapy for monogenic diseases, particularly those affecting thehematopoietic system, has provided convincing evidence that geneticcorrection of autologous hematopoietic stem cells (HSCs) is analternative therapeutic option to allogeneic HSCT, avoiding its majorcomplications (Cartier et al 2009; Cavazzana-Calvo et al 2010; Cartieret al 2012; Aiuti et al 2013; Biffi et al 2013). Genetic correction fordiseases affecting the erythrocyte such as β-thalassemia and sickle celldisease, have been addressed in animal models (Pestina et al 2009l Bredaet al 2012) and also in humans (Cavazzana-Calvo et al 2010). However,gene therapy approaches for inherited erythroid metabolic deficienciessuch as PKD are still limited. The feasibility of HSC gene therapy forPKD has been demonstrated both in mouse (Tani et al 1994; Meza et al2009) and in dog RPK deficient experimental models (Trobridge et al2012) showing that donor chimerism and transduction levels are keypoints to reach an efficient correction of the hemolytic phenotype(Richard et al 2004) given the lack of selective advantage of donorgene-corrected HSCs. Previous work with a PKD mouse model demonstratedthat retrovirally-derived human RPK expression was capable of fullycorrecting PKD phenotype when over 25% genetically corrected cells weretransplanted (Meza et al 2009). A similar therapeutic threshold ofcorrected cells was recently reported in one PKD Basenji dog infusedwith in vivo expanded and foamy vector-corrected HSCs (Trobridge et al2012).

A number of challenges remain with regard to designing polynucleotidecassettes and expression vectors for use in gene therapy. Onesignificant challenge is obtaining sufficient expression of thetransgene in target cells. A longstanding unmet need in the art has beensufficiently robust expression of transgenes following gene transfer. Insome cases, more efficient expression is required for the efficacy ofcertain vectors, for example plasmid DNA vectors. In other cases, moreefficient gene expression cassettes are desirable to allow for a lowertherapeutic dose that has a more favorable safety profile or a lessinvasive route of administration.

High levels of transgene expression can be achieved when gammaretroviral(gamma-RV) vectors are used due to the fact that the therapeutictransgene expression is regulated by their LTR sequences. However, thefirst clinical trials based on this type of vector raised safetyconcerns, as several patients developed unexpected leukemias(Hacein-Bey-Abina et al 2008) The strong promoter activity of LTRsequences could affect the regulation of surrounding genes, either byactivation of proto-oncogene promoters or by inhibition of tumoursuppressor genes, leading to insertional mutagenesis (Ott et al 2006;Howe et al 2008; Stein et al 2010; Braun et al 2014). These findingshighlighted the need to use safer and more efficient vectors than gammaretroviral vectors for the PKD gene therapy.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an expression cassettecomprising a polynucleotide sequence comprising: a) a promoter sequence;b) a sequence encoding a gene product; and c) an ribonucleic acid (RNA)export signal, wherein the promoter sequence is operably linked to thesequence encoding the pyruvate kinase polypeptide, and optionally wherea)-c) are present in the expression cassette in 5′ to 3′ order. Incertain embodiments, the promoter is a phosphoglycerate kinase (PGK)promoter. In some embodiments, the gene product is a therapeutic geneproduct. In some embodiments, the therapeutic gene product is a pyruvatekinase (PK) polypeptide, optionally a pyruvate kinase, liver and redblood cell (PKLR) polypeptide. In certain embodiments, the sequenceencoding the gene product is codon-optimized. In particular embodiments,the RNA export signal is a mutated post-transcriptional regulatoryelement of the woodchuck hepatitis virus (Wpre). In certain embodiments,the mutated Wpre is a chimeric Wpre comprising a sequence having atleast 80% identity to SEQ ID NO:1. In some embodiments, the expressioncassette further comprising one or more enhancer sequences. In someembodiments, the expression cassette further comprises a polypurinetract (PPT) or polyadenylation (polyA) signal sequence. In someembodiments, the expression cassette further comprises one or more ofthe following sequences: i) a packing signal sequence; ii) a truncatedGag sequence; iii) a Rev responsive element (RRE); iv) a centralpolypurine tract (cPPT); v) a central terminal sequence (CTS); and vi)an upstream sequence element (USE), optionally from simian virus 40(SV40-USE). In some embodiments, the expression cassette furthercomprises 5′ and 3′ long terminal repeat sequences.

In a related embodiment, the present invention provides a recombinantgene delivery vector comprising an expression cassette disclosed herein.In certain embodiments, the recombinant gene delivery vector is a virusor viral vector. In certain embodiments, the virus or viral vector is alentivirus (LV).

In another related embodiment, the present invention provides a cellcomprising an expression cassette or gene delivery vector disclosedherein. In some embodiments, the cell is a blood cell. In someembodiments, the cell is an erythroid cell. In some embodiments, thecell is a bone marrow cell, e.g., a lineage depleted bone marrow cell.In some embodiments, the cell is a hematopoietic stem cell. In someembodiments, the cell is a CD34+ hematopoietic stem cell. In someembodiments, the cell is a committed hematopoietic erythroid progenitorcell.

In yet another related embodiment, the present invention provides apharmaceutical composition comprising a pharmaceutically acceptableexcipient and recombinant gene delivery vector or cell disclosed herein.

In another embodiment, the present invention provides a method oftreating or preventing a disease or disorder in a subject in needthereof, comprising providing to the subject an expression cassette,gene delivery vector, or pharmaceutical composition disclosed herein. Inone embodiment, the disease or disorder is a Pyruvate Kinase Deficiency(PKD) and the gene product is a pyruvate kinase (PK) polypeptide,optionally a pyruvate kinase, liver and red blood cell (PKLR)polypeptide. In certain embodiments, the pharmaceutical compositioncomprises the recombinant gene delivery vector. In other embodiments,the pharmaceutical composition comprises the cell. In one embodiment,the cell is autologous to the subject. In another embodiment, the cellis allogeneic to the subject.

In a related embodiment, the present invention provides a method forexpressing a transgene in erythroid cells, comprising contacting one ormore erythroid cells with an effective amount of a recombinant viralvector, wherein the vector comprises a human phosphoglycerate kinasepromoter, a codon optimized version of a human pyruvate kinase, liverand red blood cell (PKLR) cDNA transgene, and a mutatedpost-transcriptional regulatory element of the woodchuck hepatitisvirus, wherein following said contacting, PKLR is expressed atdetectable levels in the one or more erythroid cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts a scheme reflecting the position of the differentdescribed elements present in the backbone of the lentiviral vector.

FIG. 2A is a schematic representation of the self-inactivatinglentiviral vectors used throughout gene therapy experiments harboringthe human PGK promoter regulating the expression of the EGFP transgenein the control vector (upper diagram) or the expression of acodon-optimized sequence of the PKLR gene cDNA (coRPK) in thetherapeutic vector (lower diagram).

FIG. 2b is a schematic gene therapy protocol performed to address thefunctionality of the developed PGK-coRPK lentiviral vector.

FIGS. 3a-d depict data showing correction of PKD phenotype in peripheralblood of primary recipients after genetic correction. FIG. 3a RBCs andFIG. 3b are reticulocyte levels in healthy (black bar, n=5) and PKDanemic mice (gray bar, n=6), and PKD anemic mice that were tranplantedwith the EGFP (white bar, n=9) or coRPK transduced cells (scratched bar,n=17). Data are represented as the average±SEM and were analyzed bynon-parametric Kruskal-Wallis test. FIG. 3c shows the flow cytometrystrategy used to detect the biotin labelled RBCs throughout the time andFIG. 3d RBC survival kinetics in healthy (black line, n=2), anemic (grayline, n=2) and genetically corrected mice (discontinuous line, n=4).Data are represented as the average±SEM and were analyzed by two-wayANOVA test. Healthy, non-transplanted control mice; PKD,non-transplanted PKD mice; coRPK, PKD mice expressing the therapeutictransgene.

FIGS. 4a-c show multi-lineage hematopoietic reconstitution in secondarytransplanted mice. FIG. 4a is a diagram of the flow cytometry strategyused to identify the different hematopoietic lineages by labeling withCD3-PE, B220-PE, B220-PECy5, Grl-Biotin and Mac1-Biotin antibodies plusSAV-PE-Cy5. FIG. 4b depicts representative dot-plots and FIG. 4c depictspercentages of each lineage in PB at 140 days after transplant. Barsrepresent the average percentage±SEM of healthy (n=2, black bar) and PKDmouse (n=2, grey bar) controls and secondary transplanted miceexpressing the coRPK therapeutic transgene (n=4, scratched bar).

FIGS. 5a-d depict PKD phenotype correction in secondary transplantedmice. FIG. 5a shows Brilliant Cresyl blue staining of blood smears fromnon-transplanted mice and secondary recipients to identify reticulocytepopulation (in blue). FIG. 5b is Flow cytometry analysis of reticulocytelevels in peripheral blood. FIG. 5c represents RBC percentage and FIG.5d reticulocyte percentage in secondary transplanted mice expressing thecoRPK transgene (scratched bar, n=4), in healthy mice (black bar, n=3)and in anemic control mice (grey bar, n=3). Data are represented theaverage±SEM and were analyzed by non-parametric two-tailed Mann-Whitneytest.

FIGS. 6a-c show quantification of proviral integrations. FIG. 6a showsvector copy number per cell in BM CFUs from individual transplanted miceat 120 to 170 days after transplant. Transduction and chimerismpercentages are also shown. FIG. 6b shows provirus copy number in cellsfrom different hematopoietic compartments. Columns represent theaverage±SEM of the different groups of transplanted mice. FIG. 6c showsthe kinetics of proviral integrations in BM cells from individualtransplanted EGFP-expressing mice (grey lines) and mice carrying thecoRPK transgene (black lines).

FIGS. 7a-c depict the normalization of the erythroid differentiationpattern in genetically corrected mice. FIG. 7a shows percentages of thedifferent erythroid subpopulation in bone marrow and spleen at 140 daysafter transplant. FIG. 7b depicts representative dot plots of the flowcytometry strategy used. The expression intensity of the CD71 and Ter119markers allows for identifying four erythroid subpopulations. populationI: early proerythroblasts (Ter119^(med) CD71^(high)), population II:basophilic erythroblasts (Ter119^(high) CD71^(high)), population III:late basophilic and polychromatophilic erythroblasts (Ter119^(high)CD71^(med)) and population IV: orthochromatophilic erythroblasts,reticulocytes and mature erythroid cells (Ter119^(high) CD71^(low)).FIG. 7c shows plasma Epo levels measured by ELISA in non-transplantedand transplanted mice. Dots represent values of individual mice. Linesrepresent average±SEM and were analyzed by non-parametric Kruskal-Wallistest. Healthy, non-transplanted control mice; PKD, non-transplanted PKDmice; EGFP, PKD mice expressing the EGFP transgene; coRPK, PKD miceexpressing the therapeutic transgene.

FIGS. 8a-b show hematopoietic progenitor assays in control mice andtransplanted mice with transduced cells. The data demonstrate total CFUsfrom spleen (FIG. 8a ) and bone marrow (FIG. 8b ) at 140 days aftertransplant. Dots represent number of colonies per mouse analyzed andlines represent average±SEM in each group. Data were statisticallyanalyzed by non-parametric Kruskal-Wallis test.

FIGS. 9a-c demonstrate reversion of splenomegaly and organ pathology ingenetically corrected mice at 140 days post-transplantation. FIG. 9a arepictures of representative spleens and FIG. 9b shows ratio of spleenweight to total body weight from primary and secondary transplanted PKDmice. Dots represent values of individual mice. Lines representaverage±SEM per group. Data were analyzed by non-parametricKruskal-Wallis test. FIG. 9c shows the histological study of spleen andliver from primary transplanted PKD mice. First and second column showthe representative histology sections of spleen and liver stained withhematoxylin-eosin and photographed using a 4× and 10× objective,respectively, in a light microscope. Arrows point to erythroid cellclusters indicative of extramedullary erythropoiesis. Third column showsPrussian blue staining (Fe) of liver sections to detect iron depositsindicated by arrowheads. Photographs were taken using a 20× objective.Group legends as in FIG. 7. 2^(nd) coRPK, secondary recipients.

FIGS. 10a-g depict metabolic profiling in RBC samples from micetransplanted with genetically modified cells. Analysis of significantmetabolic profile changes in healthy and transplanted mice by comparisonto PKD animals in two independent experiments. FIG. 10a shows thecomplete RBC heat map obtained by untargeted profiling, where higher andlower metabolite levels are represented in red and blue respectively.Metabolites listed have at least one comparison that is significantusing the following criteria: absolute fold change>1.5; minimalsignal>2000; Adjusted p-value<0.01. Black boxes highlight cluster ofmetabolite changes with distinct profile among the groups. FIGS. 10b,10c, and 10d depict ATP, ADP and pyruvate levels in RBCs, respectively,measured by untargeted profiling by comparison to PKD mice at 140 daysafter transplant. Assay 1: Healthy mice (black bars) n=1, PKD (greybars) n=1, hPGK-EGFP (white bars) n=2, hPGK-coRPK (scratched bars) n=3.Assay 2: Healthy mice n=2, PKD n=2, hPGK-EGFP n=6, hPGK-coRPK n=10.FIGS. 10e, 10f, and 10g depict RBC targeted metabolic profiling of aselected number of metabolites involved in the glycolytic pathway (PEP,3-phosphoglyceric acid and D-lactic acid, respectively) at 280 dayspost-transplantation. Dots represent values of individual mice. Linesrepresent average±SEM and were analyzed by non-parametric Kruskal-Wallistest. Assay 2: Healthy mice n=7, PKD n=5, hPGK-EGFP n=3, hPGK-coRPK n=5.

FIGS. 11a-c depict pyruvate Kinase activity, FIG. 11b hexokinaseactivity and FIG. 11c ratio of Pyruvate Kinase and Hexokinase enzymaticactivities in RBCs from control mice and mice transplanted withtransduced cells. RBCs were purified from blood samples through acellulose column to avoid leukocyte PK activity contamination andsubjected to enzyme activity evaluation. Black bars, healthy mice (n=2);white bars, mice transplanted with cells transduced with the EGFPexpressing vector (n=3); scratched bars, mice transplanted with cellstransduced with the coRPK expressing vector (n=3). Checkered barsrepresent values from a healthy volunteer (n=1). Data represent theaverage±SEM of each group.

FIGS. 12a-d show untargeted metabolic profiling in WBC samples from micetransplanted with genetically modified cells. FIG. 12a represents theprincipal component analysis of untargeted metabolite profile in RBCs(red dots; left and center) and WBCs (blue dots; cluster on right) incontrol and transplanted mice. FIGS. 12b, 12c, and 12d depict ATP, ADPand pyruvate levels, respectively, in WBCs by comparison to PKD mice.Assay 1: healthy mice (black bars) n=1, PKD (grey bars) n=1, hPGK-EGFP(white bars) n=2, hPGK-coRPK (scratched bars) n=3. Assay 2: healthy micen=2, PKD n=2, hPGK-EGFP n=6, hPGK-coRPK n=10. Data represent theaverage±SEM per group and were analyzed by non-parametric Kruskal-Wallistest.

FIG. 13 shows a gel image of LAM-PCR products generated with Tsp5091enzyme for samples harvested from all mice at different time points andtissues. Vector integration sites were identified by LAM-PCRamplification of 3′vector LTR-genome junctions. A MultiNA automatedsystem was used, generating a pattern characterized by several bands.Vector backbone derived Tsp5091 internal control band (IC) is indicatedby an arrow.

FIG. 14 shows a gel image of LAM-PCR products generated with HpyCH4IV5enzyme for samples harvested from all mice at different time points andtissues. Vector integration sites were identified by LAM-PCRamplification of 3′ vector LTR-genome junctions. A MultiNA automatedsystem was used, generating a pattern characterized by several bands.Vector backbone derived HpyCH4IV5 internal control band (IC) isindicated by an arrow.

FIG. 15 depicts a general scheme of the analysis of integration sitemapping performed in mice transplanted with genetically modifiedhematopoietic progenitors. Bone marrow and white blood cell samples fromtransplanted mice belonging to two independent experiments (Table 3) andharvested at different time-points after transplant were analyzed asdescribed in supplementary methods following the showed pipeline.

FIGS. 16a-b show the distribution of LV integrations along the genome oftransplanted mice. FIG. 16a depicts Integration site (IS) frequencydistribution around Transcription Start Site (TSS) of the nearest RefSeqgene, spanning 500 Kb upstream and downstream the TSS. Numbers on thetop are the number of IS detected for all samples and time-points. FIG.16b depicts chromosomal distribution of LV integration sites intransplanted mice expressing the EGFP transgene (black bars) or thecoRPK therapeutic transgene (grey bars), showing no skewing towards anyparticular chromosome.

FIGS. 17a-c demonstrates clonal abundance analysis of coRPK-LVtransduced cells. Dots plot representation of clonal abundance of pooledintegrations in each mouse from assays 1 (FIG. 17a ) and 2 (FIGS. 17band 17c ). The relative percentage (y-axis) for each integration site isrelative to the total number of sequences reads obtained in eachdataset. IS, integration site; BM, bone marrow; PB, peripheral blood;coRPK1-14, mice transplanted with hematopoietic cells transduced withthe therapeutic vector.

FIG. 18 presents the tracked shared integrations between primary andsecondary recipient mice carrying the therapeutic PGK-coRPK LV vector.Integrations detected in either mouse in any organ and at any time arepooled. Secondary recipients received the pooled BM from transplantedmice coRPK11 to 14. The rest of the IS detected were detected or in theprimary or in the secondary recipients. Numbers in the boxes show therepresentativeness in percentage of the corresponding integration in thereferred mouse. In addition to >5% filter applied on integrationanalysis, all integration with a sequence count<3 were eliminated.

FIG. 19 demonstrates clonal abundance analysis of EGFP-LV transducedcells. Dots plot representation of clonal abundance of pooledintegrations in each mouse in bone marrow. The relative percentage(y-axis) for each integration site is relative to the total number ofsequences reads obtained in each dataset. Similarly to co-RPK transducedcells (FIG. 17), the graph indicates that the vast majority oftransplanted mice show a polyclonal pattern of hematopoieticrepopulation. IS, Integration site

FIG. 20 depicts the LV genomic integration profile. Gene Ontology (GO)analysis was performed using the GREAT software on samples fromtransplanted mouse. All integrations retrieved from this study (N=2220)showed overrepresentations of the gene functions indicated on the leftpart of the figure. To address if the most abundant integrations wereenriched on specific gene classes, all integration sites with a relativesequence count>5% of the entire dataset (shown in FIG. 17) wereselected, showing no GO gene classes overrepresented.

FIG. 21 depicts a schematic diagram of the medicinal product (PGK-coRPKLV).

FIGS. 22a-b depicts the mechanism of action of the medicinal product.FIG. 22a depicts the ectopic expression of the PGK-coRPK LV medicinalproduct will rescue the wild-type phenotype of PKD erythrocytes,otherwise unable to generate a functional RPK protein to producesufficient energy to carry out their functions. FIG. 22b shows the genetherapy strategy for PKD patients based on the ex vivo transduction ofRPK deficient CD34⁺ hematopoietic progenitors with the medicinal productand subsequent transplantation into the patient. The developed medicinalproduct carrying the therapeutic human PKLR gene cDNA will be integratedin the patient CD34⁺ cell genome upon ex vivo transduction. Thesegenetically corrected cells will be then infused back into the patient,where they will produce RBCs expressing the therapeutic transgene, andtherefore, producing functional RPK proteins that will correct the PKDpathological phenotype. Figure modified from the Boston Children'sHospital blog.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell.Illustrative vectors include, for example, plasmids, viral vectors,liposomes, and other gene delivery vehicles.

The term “LV” is an abbreviation for lentivirus, and may be used torefer to the virus itself or derivatives thereof. The term covers allsubtypes and both naturally occurring and recombinant forms, exceptwhere required otherwise.

As used herein, the term “gene” or “coding sequence” refers to anucleotide sequence in vitro or in vivo that encodes a gene product. Insome instances, the gene consists or consists essentially of codingsequence, that is, sequence that encodes the gene product. In otherinstances, the gene comprises additional, non-coding, sequence. Forexample, the gene may or may not include regions preceding and followingthe coding region, e.g. 5′ untranslated (5′ UTR) or “leader” sequencesand 3′ UTR or “trailer” sequences, as well as intervening sequences(introns) between individual coding segments (exons).

As used herein, a “therapeutic gene” refers to a gene that, whenexpressed, confers a beneficial effect on the cell or tissue in which itis present, or on a mammal in which the gene is expressed. Examples ofbeneficial effects include amelioration of a sign or symptom of acondition or disease, prevention or inhibition of a condition ordisease, or conferral of a desired characteristic. Therapeutic genesinclude genes that correct a genetic deficiency in a cell or mammal.

As used herein, a transgene is a gene that is delivered to a cell by avector.

As used herein, the term “gene product” refers to the desired expressionproduct of a polynucleotide sequence such as a polypeptide, peptide,protein or interfering RNA including short interfering RNA (siRNA),miRNA or small hairpin RNA (shRNA).

As used herein, the terms “polypeptide,” “peptide,” and “protein” referto polymers of amino acids of any length. The terms also encompass anamino acid polymer that has been modified; for example, disulfide bondformation, glycosylation, lipidation, phosphorylation, or conjugationwith a labeling component.

By “comprising” it is meant that the recited elements are required in,for example, the composition, method, kit, etc., but other elements maybe included to form the, for example, composition, method, kit etc.within the scope of the claim. For example, an expression cassette“comprising” a gene encoding a therapeutic polypeptide operably linkedto a promoter is an expression cassette that may include other elementsin addition to the gene and promoter, e.g. poly-adenylation sequence,enhancer elements, other genes, linker domains, etc.

By “consisting essentially of”, it is meant a limitation of the scope ofthe, for example, composition, method, kit, etc., described to thespecified materials or steps that do not materially affect the basic andnovel characteristic(s) of the, for example, composition, method, kit,etc. For example, an expression cassette “consisting essentially of” agene encoding a therapeutic polypeptide operably linked to a promoterand a polyadenylation sequence may include additional sequences, e.g.linker sequences, so long as they do not materially affect thetranscription or translation of the gene. As another example, a variant,or mutant, polypeptide fragment “consisting essentially of” a recitedsequence has the amino acid sequence of the recited sequence plus orminus about 10 amino acid residues at the boundaries of the sequencebased upon the full length naïve polypeptide from which it was derived,e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue less than the recitedbounding amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10residues more than the recited bounding amino acid residue.

By “consisting of”, it is meant the exclusion from the composition,method, or kit of any element, step, or ingredient not specified in theclaim. For example, an expression cassette “consisting of” a geneencoding a therapeutic polypeptide operably linked to a promoter, and apost-transcriptional regulatory element consists only of the promoter,polynucleotide sequence encoding the therapeutic polypeptide, andpost-transcriptional regulatory element. As another example, apolypeptide “consisting of” a recited sequence contains only the recitedsequence.

An “expression vector” as used herein encompasses a vector, e.g.plasmid, minicircle, viral vector, liposome, and the like as discussedabove or as known in the art, comprising a polynucleotide which encodesa gene product of interest, and is used for effecting the expression ofa gene product in an intended target cell. An expression vector alsocomprises control elements operatively linked to the encoding region tofacilitate expression of the gene product in the target. The combinationof control elements, e.g. promoters, enhancers, UTRs, miRNA targetingsequences, etc., and a gene or genes to which they are operably linkedfor expression is sometimes referred to as an “expression cassette.”Many such control elements are known and available in the art or can bereadily constructed from components that are available in the art.

A “promoter” as used herein encompasses a DNA sequence that directs thebinding of RNA polymerase and thereby promotes RNA synthesis, i.e., aminimal sequence sufficient to direct transcription. Promoters andcorresponding protein or polypeptide expression may be ubiquitous,meaning strongly active in a wide range of cells, tissues and species orcell-type specific, tissue-specific, or species specific. Promoters maybe “constitutive,” meaning continually active, or “inducible,” meaningthe promoter can be activated or deactivated by the presence or absenceof biotic or abiotic factors. Also included in the nucleic acidconstructs or vectors of the invention are enhancer sequences that mayor may not be contiguous with the promoter sequence. Enhancer sequencesinfluence promoter-dependent gene expression and may be located in the5′ or 3′ regions of the native gene.

An “enhancer” as used herein encompasses a cis-acting element thatstimulates or inhibits transcription of adjacent genes. An enhancer thatinhibits transcription also is termed a “silencer”. Enhancers canfunction (i.e., can be associated with a coding sequence) in eitherorientation, over distances of up to several kilobase pairs (kb) fromthe coding sequence and from a position downstream of a transcribedregion.

A “termination signal sequence” as used herein encompasses any geneticelement that causes RNA polymerase to terminate transcription, such asfor example a polyadenylation signal sequence.

As used herein, the terms “operatively linked” or “operably linked”refers to a juxtaposition of genetic elements, e.g. promoter, enhancer,termination signal sequence, polyadenylation sequence, etc., wherein theelements are in a relationship permitting them to operate in theexpected manner. For instance, a promoter is operatively linked to acoding region if the promoter helps initiate transcription of the codingsequence. There may be intervening residues between the promoter andcoding region so long as this functional relationship is maintained.

As used herein, the term “heterologous” means derived from agenotypically distinct entity from that of the rest of the entity towhich it is being compared. For example, a polynucleotide introduced bygenetic engineering techniques into a plasmid or vector derived from adifferent species is a heterologous polynucleotide. As another example,a promoter removed from its native coding sequence and operativelylinked to a coding sequence with which it is not naturally found linkedis a heterologous promoter. Thus, for example, an LV vector thatincludes a heterologous nucleic acid encoding a heterologous geneproduct is an LV vector that includes a nucleic acid not normallyincluded in a naturally-occurring, wild-type LV, and the encodedheterologous gene product is a gene product not normally encoded by anaturally-occurring, wild-type LV.

The term “endogenous” as used herein with reference to a nucleotidemolecule or gene product refers to a nucleic acid sequence, e.g. gene orgenetic element, or gene product, e.g. RNA, protein, that is naturallyoccurring in or associated with a host virus or cell.

The term “native” as used herein refers to a nucleotide sequence, e.g.gene, or gene product, e.g. RNA, protein, that is present in a wildtypevirus or cell.

The term “variant” as used herein refers to a mutant of a referencepolynucleotide or polypeptide sequence, for example a nativepolynucleotide or polypeptide sequence, i.e. having less than 100%sequence identity with the reference polynucleotide or polypeptidesequence. Put another way, a variant comprises at least one amino aciddifference (e.g., amino acid substitution, amino acid insertion, aminoacid deletion) relative to a reference polynucleotide sequence, e.g. anative polynucleotide or polypeptide sequence. For example, a variantmay be a polynucleotide having a sequence identity of 70% or more with afull length native polynucleotide sequence, e.g. an identity of 75% or80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99%identity with the full length native polynucleotide sequence. As anotherexample, a variant may be a polypeptide having a sequence identity of70% or more with a full length native polypeptide sequence, e.g. anidentity of 75% or 80% or more, such as 85%, 90%, or 95% or more, forexample, 98% or 99% identity with the full length native polypeptidesequence. Variants may also include variant fragments of a reference,e.g. native, sequence sharing a sequence identity of 70% or more with afragment of the reference, e.g. native, sequence, e.g. an identity of75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98%or 99% identity with the native sequence.

As used herein, the terms “biological activity” and “biologicallyactive” refer to the activity attributed to a particular biologicalelement in a cell. For example, the “biological activity” of an“immunoglobulin”, “antibody” or fragment or variant thereof refers tothe ability to bind an antigenic determinant and thereby facilitateimmunological function. As another example, the biological activity of apolypeptide or functional fragment or variant thereof refers to theability of the polypeptide or functional fragment or variant thereof tocarry out its native functions of, e.g., binding, enzymatic activity,etc. As a third example, the biological activity of a gene regulatoryelement, e.g. promoter, enhancer, kozak sequence, and the like, refersto the ability of the regulatory element or functional fragment orvariant thereof to regulate, i.e. promote, enhance, or activate thetranslation of, respectively, the expression of the gene to which it isoperably linked.

The terms “administering” or “introducing”, as used herein, refer todelivery of a vector for recombinant protein expression to a cell, tocells and/or organs of a subject, or to a subject. Such administering orintroducing may take place in vivo, in vitro or ex vivo. A vector forexpression of a gene product may be introduced into a cell bytransfection, which typically means insertion of heterologous DNA into acell by physical means (e.g., calcium phosphate transfection,electroporation, microinjection or lipofection); infection, whichtypically refers to introduction by way of an infectious agent, i.e. avirus; or transduction, which typically means stable infection of a cellwith a virus or the transfer of genetic material from one microorganismto another by way of a viral agent (e.g., a bacteriophage).

“Transformation” is typically used to refer to bacteria comprisingheterologous DNA or cells which express an oncogene and have thereforebeen converted into a continuous growth mode such as tumor cells. Avector used to “transform” a cell may be a plasmid, virus or othervehicle.

Typically, a cell is referred to as “transduced”, “infected”;“transfected” or “transformed” dependent on the means used foradministration, introduction or insertion of heterologous DNA (i.e., thevector) into the cell. The terms “transduced”, “transfected” and“transformed” may be used interchangeably herein regardless of themethod of introduction of heterologous DNA.

The term “host cell”, as used herein refers to a cell which has beentransduced, infected, transfected or transformed with a vector. Thevector may be a plasmid, a viral particle, a phage, etc. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art. It will be appreciated that the term “hostcell” refers to the original transduced, infected, transfected ortransformed cell and progeny thereof.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof, e.g. reducing thelikelihood that the disease or symptom thereof occurs in the subject,and/or may be therapeutic in terms of a partial or complete cure for adisease and/or adverse effect attributable to the disease. “Treatment”as used herein covers any treatment of a disease in a mammal, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;or (c) relieving the disease, i.e., causing regression of the disease.The therapeutic agent may be administered before, during or after theonset of disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy will desirably be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, human and non-human primates, including simians and humans;mammalian sport animals (e.g., horses); mammalian farm animals (e.g.,sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents(e.g., mice, rats, etc.).

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art and the practice of the presentinvention will employ conventional techniques of microbiology andrecombinant DNA technology, which are within the knowledge of those ofskill of the art.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of cell biology, molecular biology(including recombinant techniques), microbiology, biochemistry andimmunology, which are within the scope of those of skill in the art.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M.Weir & C. C. Blackwell, eds.); “Gene Transfer Vectors for MammalianCells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR: ThePolymerase Chain Reaction”, (Mullis et al., eds., 1994); and “CurrentProtocols in Immunology” (J. E. Coligan et al., eds., 1991), each ofwhich is expressly incorporated by reference herein.

In certain embodiments, the present disclosure provides polynucleotides,polynucleotide cassettes and expression vectors for the expression of agene in cells. Also provided are pharmaceutical compositions and methodsfor the use of any of the compositions in promoting the expression of agene in cells, for example, in an individual, e.g. for the treatment orprophylaxis of a disorder. These and other objects, advantages, andfeatures of the invention will become apparent to those persons skilledin the art upon reading the details of the compositions and methods asmore fully described below.

The present invention relates generally to the fields of molecularbiology and virology, and in particular, to genetic expressioncassettes, and vectors comprising them useful for the delivery ofnucleic acid segments encoding selected therapeutic constructs(including for example, peptides, polypeptides, ribozymes, and catalyticRNA molecules), to selected cells and tissues of vertebrate animals. Inparticular, these genetic constructs are useful in the development ofgene therapy vectors, including for example, lentiviral vectors, for thetreatment of mammalian, and in particular, human, diseases, disorders,and dysfunctions.

The disclosed compositions may be utilized in a variety ofinvestigative, diagnostic and therapeutic regimens, including theprevention and treatment of a variety of human diseases. The variouscompositions and methods of the invention are described below.

Although particular compositions and methods are exemplified herein, itis understood that any of a number of alternative compositions andmethods are applicable and suitable for use in practicing the invention.It will also be understood that an evaluation of the expressionconstructs and methods of the invention may be carried out usingprocedures standard in the art.

In certain embodiments, methods and compositions are provided forpreparation of gene therapy vector compositions, e.g., viral vectors,comprising these genetic expression cassettes for use in the preparationof medicaments useful in central and targeted gene therapy of diseases,disorders, and dysfunctions in an animal, and in humans in particular.

In some embodiments, the present invention provides for gene therapy forPKD based on a lentiviral vector harbouring the hPGK eukaryotic promoterthat drives the expression of the PKLR cDNA. This therapeutic vector maybe used to transduce mouse PKD hematopoietic stem cells (HSCs) andsubsequently transplanted into myeloablated PKD mice. Ectopic RPKexpression normalizes the erythroid compartment correcting thehematological phenotype and reverting organ pathology. Metabolomicstudies demonstrate functional correction of the glycolytic pathway inRBCs derived from genetically corrected PKD HSCs, with no metabolicdisturbances in leukocytes. The analysis of the lentiviral insertionsites in the genome of transplanted hematopoietic cells demonstrates noevidence of genotoxicity in any of the transplanted animals. Overall,the results underscore the therapeutic potential of the hPGK-coRPKlentiviral vector and provide high expectations towards the gene therapyof PKD and other erythroid metabolic genetic disorders.

In certain embodiments, the present invention provides an RPK lentiviralvector (LV) for the genetic correction of PKD. Genetic modification ofmurine PKD-HSCs with this vector can efficiently correct the hemolyticphenotype and the RBC metabolite profile in transplanted PKD mice.Remarkably, no evidence of metabolic disturbances in leukocytes andgenotoxicity derived from the vector integration are observed,supporting the therapeutic potential of the PGK-coRPK LV vector.Overall, results provide encouraging evidence of the feasibility of genetherapy for PKD with a LV designed for clinical application.

Certain embodiments of the present invention comprise aself-inactivating lentiviral vector expressing a codon-optimized versionof human PKLR gene. The expression vector comprises a promoter region, acoding sequence, and a post-transcriptional regulatory element.

Certain embodiments of polynucleotide cassettes of the present inventioncomprise a promoter region comprising a promoter sequence, or afunctional fragment thereof. In one embodiment, the promoter is a humanphosphoglycerate kinase (PGK) promoter.

Some embodiments of the present invention comprise polynucleotidecassettes for the enhanced expression of pyruvate kinase. In someembodiments, the polynucleotide cassette comprises a codon-optimizedversion of the human PKLR cDNA (coRPK) to increase mRNA stability upontranscription. For the optimization, GeneArt® software may be used,increasing the GC content and removing cryptic splice sites in order toavoid transcriptional silencing and therefore increase transgeneexpression. The coRPK optimized sequence showed 80.4% homology with thehuman PKLR gene, with no changes in the amino acids sequence of theprotein. Alternatively, any optimization method known in the art may beused.

In some embodiments, the polynucleotide cassette comprises an RNA exportsignal downstream of the second enhancer. The RNA export signal maycomprise woodchuck hepatitis virus post-transcriptional element (WPRE)sequence. In some embodiments, a mutated post-transcriptional regulatoryelement of the woodchuck hepatitis virus (Wpre), lacking any residualopen reading frame (Schambach, Bohne et al. 2006) is also included toimprove the level of expression and stability of the therapeutic gene.

In some aspects of the invention, gene delivery vectors are providedcomprising a polynucleotide cassette of the present invention. In someembodiments, the gene delivery vector is a lentivirus.

In some aspects of the invention, pharmaceutical compositions areprovided comprising a polynucleotide cassette of the invention and apharmaceutical excipient. In some embodiments, the pharmaceuticalcomposition comprises a gene delivery vector of the invention and apharmaceutical excipient.

In some aspects of the invention, methods are provided for expressing atransgene in mammalian cells. In some embodiments, the method comprisescontacting one or more mammalian cells with an effective amount of apolynucleotide cassette of the invention or a gene delivery vector ofthe invention, wherein the transgene is expressed at detectable levelsin the one or more mammalian cells. In some embodiments, the methodcomprises contacting one or more mammalian cells with an effectiveamount of a polynucleotide cassette of the invention or a gene deliveryvector of the invention, wherein the transgene is expressed attherapeutic levels in the one or more mammalian cells. In someembodiments, the method is in vitro. In other embodiments, the method isin vivo.

In some aspects of the invention, methods are provided for the treatmentor prophylaxis of a disease or disorder in a mammal in need of treatmentor prophylaxis for a disease or disorder. In some embodiments, themethod comprises administering to the mammal an effective amount of apharmaceutical composition of the invention, wherein the coding sequenceencodes a therapeutic gene product.

Compositions

In some aspects of the disclosure, compositions are provided for theexpression of a transgene in a eukaryotic cell(s). In some aspects, theeukaryotic cell is a mammalian cell. In some aspects, the mammalian cellis a hematopoietic stem cell. In some embodiments, the cell is a bonemarrow cell, e.g., a lineage depleted bone marrow cell. In some aspects,the mammalian cell is a committed hematopoietic erythroid progenitor.

In some embodiments of the disclosure, the composition is apolynucleotide cassette. By a “polynucleotide cassette” is meant apolynucleotide sequence comprising two or more functional polynucleotidesequences, e.g. regulatory elements, translation initiation sequences,coding sequences, and/or termination sequences, etc., typically inoperable linkage to one another. Likewise, by a “polynucleotide cassettefor the expression of a transgene in a mammalian cell,” it is meant acombination of two or more functional polynucleotide sequences, e.g.promoter, enhancer, 5′UTR, translation initiation sequence, codingsequence, and/or termination sequences, etc. that promotes theexpression of the transgene in a cell.

For example, in some embodiments, the polynucleotide cassette comprises:human phosphoglycerate kinase (PGK) promoter, a codon-optimized versionof the human PKLR cDNA (coRPK), and a mutated post-transcriptionalregulatory element of the woodchuck hepatitis virus (Wpre).

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the human PKLR promoter comprises orconsists of the following sequence, a functional fragment thereof, or asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identity to the following sequence:

(SEQ ID NO: 4) ATTATGGTAAATCCACTTACTGTCTGCCCTCGTAGCCATCGAGATAAACCCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCT TTCGACCTGCAGCCC.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the human PKLR promoter comprises orconsists of the following sequence, a functional fragment thereof, or asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identity to the following sequence:

(SEQ ID NO: 8) TCCACGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTCGTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGGCGCCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCAC CGACCTCTCTCCCCAG.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the RPE sequence comprises orconsists of the following sequence, or a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identity to the following sequence:

(SEQ ID NO: 3) TCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGA TACCT.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the psi sequence is an HIV-1 psisequence or the psi sequence comprises or consists of the followingsequence, or a sequence having at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identity to the followingsequence:

(SEQ ID NO: 5) TCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAG.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the 5′ LTR comprises or consists ofthe following sequence, or a sequence having at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, or at least 99% identity tothe following sequence:

(SEQ ID NO: 6) TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the 3′ LTR comprises or consists ofthe following sequence, or a sequence having at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, or at least 99% identity tothe following sequence:

(SEQ ID NO: 7) TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG.

In some embodiments, the polynucleotide cassettes of the presentdisclosure provide for enhanced expression of a transgene in mammaliancells. As demonstrated by the working examples of the presentdisclosure, the present inventors have discovered a number ofpolynucleotide elements, i.e. improved elements as compared to thoseknown in the art, which individually and synergistically provide for theenhanced expression of transgenes in mammalian cells. In certainembodiments, the arrangement of the two or more functionalpolynucleotide sequences within the polynucleotide cassettes of thepresent disclosure provide for enhanced expression of a transgene inmammalian cells. By “enhanced” it is meant that expression of thetransgene is increased, augmented, or stronger, in cells carrying thepolynucleotide cassettes of the present disclosure relative to in cellscarrying the transgene operably linked to comparable regulatoryelements, e.g. as known in the art. Put another way, expression of thetransgene is increased, augmented, or stronger, from the polynucleotidecassettes of the present disclosure relative to expression from apolynucleotide cassette not comprising the one or more optimizedelements of the present disclosure, i.e. a reference control. In certainembodiment, the enhanced expression is specific for or limited to one ormore desired cell types.

For example, expression of the transgene may be enhanced, or augmented,or stronger, in cells comprising a polynucleotide cassette comprising apromoter disclosed herein than in cells that carry the transgeneoperably linked to a different promoter, e.g. as known in the art. Asanother example, expression of the transgene may be enhanced, orincreased, augmented, or stronger, in cells comprising a polynucleotidecassette comprising an enhancer sequence disclosed herein than in cellsthat carry the transgene operably linked to a different enhancersequence.

Promoter and enhancer elements can be tissue specific or stage-specific.For example, a tissue-specific promoter or enhancer preferentiallydrives expression (or a higher level of expression) in one or moreparticular cell type. Examples of cell types include but are not limitedto: hematopoietic stem cells, long term hematopoietic stem cells, shortterm hematopoietic stem cells, multipotent progenitors, hematopoieticCD34+ cells and any cluster differentiation subpopulation within theCD34+ population. A stage-specific promoter or enhancer preferentiallydrives expression (or higher level of expression) during one or morespecific stages of the cell cycle or development. These include but arenot limited to beta-globin locus control region, spectrin promoter, andan erythroid specific promote.

Without wishing to be bound by theory, enhanced expression of atransgene in cells is believed to be due to a faster build-up of geneproduct in the cells or a more stable gene product in the cells. Thus,enhanced expression of a transgene by the polynucleotide cassettes ofthe subject disclosure may be observed in a number of ways. For example,enhanced expression may be observed by detecting the expression of thetransgene following contact of the polynucleotide cassette to the cellssooner, e.g. 2 days sooner, 7 days sooner, 2 weeks sooner, 3 weekssooner, 4 weeks sooner, 8 weeks sooner, 12 weeks sooner or more, thanexpression would be detected if the transgene were operably linked tocomparable regulatory elements, e.g. as known in the art. Enhancedexpression may also be observed as an increase in the amount of geneproduct per cell. For example, there may be a 2-fold increase or more,e.g. a 3-fold increase or more, a 4-fold increase or more, a 5-foldincrease or more, or a 10-fold increase or more in the amount of geneproduct per mammalian cell. Enhanced expression may also be observed asan increase in the number of mammalian cells that express detectablelevels of the transgene carried by the polynucleotide cassette. Forexample, there may be a 2-fold increase or more, e.g. a 3-fold increaseor more, a 4-fold increase or more, a 5-fold increase or more, or a10-fold increase or more in the number of mammalian cells that expressdetectable levels of the transgene. As another example, thepolynucleotide of the present invention may promote detectable levels ofthe transgene in a greater percentage of cells as compared to aconventional polynucleotide cassette; for example, where a conventionalcassette may promote detectable levels of transgene expression in, forexample, less than 5% of the cells in a certain region, thepolynucleotide of the present invention promotes detectable levels ofexpression in 5% or more of the cells in that region; e.g. 10% or more,15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% ormore, or 45% or more, in some instances 50% or more, 55% or more; 60% ormore, 65% or more, 70% or more, or 75% or more, for example 80% or more,85% or more, 90% or more, or 95% or more of the cells that arecontacted, will express detectable levels of gene product. Enhancedexpression may also be observed as an alteration in the viability and/orfunction of the cells.

The polynucleotide cassettes of the present disclosure typicallycomprise a promoter region. Any suitable promoter region or promotersequence therein can be used in the subject polynucleotide cassettes, solong as the promoter region promotes expression of a coding sequence ineukaryotic cells. In certain embodiments, the promoter region promoterexpression of a coding sequence in mammalian cells. In some instances,the promoter is a ubiquitous promoter, i.e., it is a promoter that isactive in a wide range of cells, tissues and species. In otherinstances, the promoter is a human phosphoglycerate kinase (PGK)promoter.

In some embodiments, the polynucleotide comprises one or more enhancers.Enhancers are nucleic acid elements known in the art to enhancetranscription, and can be located anywhere in association with the genethey regulate, e.g. upstream, downstream, within an intron, etc. Anyenhancer element can be used in the polynucleotide cassettes and genetherapy vectors of the present disclosure, so long as it enhancesexpression of the gene when used in combination with the promoter.

The coding sequence to be expressed in the cells can be anypolynucleotide sequence, e.g. gene or cDNA that encodes a gene product,e.g. a polypeptide or RNA-based therapeutic (siRNA, antisense, ribozyme,shRNA, etc.). The coding sequence may be heterologous to the promotersequence to which it is operably linked, i.e. not naturally operablyassociated with it. Alternatively, the coding sequence may be endogenousto the promoter sequence to which it is operably linked, i.e. isassociated in nature with that promoter. The gene product may actintrinsically in the mammalian cell, or it may act extrinsically, e.g.it may be secreted. For example, when the transgene is a therapeuticgene, the coding sequence may be any gene that encodes a desired geneproduct or functional fragment or variant thereof that can be used as atherapeutic for treating a disease or disorder. In various preferredembodiments, the transgene encodes human PKLR.

In one embodiment of the invention, the transgene coding sequence ismodified, or “codon optimized” to enhance expression by replacinginfrequently represented codons with more frequently represented codons.The coding sequence is the portion of the mRNA sequence that encodes theamino acids for translation. During translation, each of 61trinucleotide codons are translated to one of 20 amino acids, leading toa degeneracy, or redundancy, in the genetic code. However, differentcell types, and different animal species, utilize tRNAs (each bearing ananticodon) coding for the same amino acids at different frequencies.When a gene sequence contains codons that are infrequently representedby the corresponding tRNA, the ribosome translation machinery may slow,impeding efficient translation. Expression can be improved via “codonoptimization” for a particular species, where the coding sequence isaltered to encode the same protein sequence, but utilizing codons thatare highly represented, and/or utilized by highly expressed humanproteins (Cid-Arregui et al., 2003; J. Virol. 77: 4928). In one aspectof the present invention, the coding sequence of the transgene ismodified to replace codons infrequently expressed in mammal or inprimates with codons frequently expressed in primates. For example, insome embodiments, the coding sequence encoded by the transgene encodes apolypeptide having at least 85% sequence identity to a polypeptideencoded by a sequence disclosed above or herein, for example at least90% sequence identity, e.g. at least 95% sequence identity, at least 98%identity, at least 99% identity, wherein at least one codon of thecoding sequence has a higher tRNA frequency in humans than thecorresponding codon in the sequence disclosed above or herein.

In an additional embodiment of the invention, the transgene codingsequence is modified to enhance expression by termination or removal ofopen reading frames (ORFs) that do not encode the desired transgene. Anopen reading frame (ORF) is the nucleic acid sequence that follows astart codon and does not contain a stop codon. ORFs may be in theforward or reverse orientation, and may be “in frame” or “out of frame”compared with the gene of interest. Such open reading frames have thepotential to be expressed in an expression cassette alongside the geneof interest, and could lead to undesired adverse effects. In one aspectof the present invention, the coding sequence of the transgene has beenmodified to remove open reading frames by further altering codon usage.This was done by eliminating start codons (ATG) and introducing stopcodons (TAG, TAA, or TGA) in reverse orientation or out-of-frame ORFs,while preserving the amino acid sequence and maintaining highly utilizedcodons in the gene of interest (i.e., avoiding codons withfrequency<20%). In the present invention, the transgene coding sequencemay be optimized by either of codon optimization and removal ofnon-transgene ORFs or using both techniques. As will be apparent to oneof ordinary skill in the art, it is preferable to remove or minimizenon-transgene ORFs after codon optimization in order to remove ORFsintroduced during codon optimization.

In some embodiments, the polynucleotide cassette of the presentinvention further comprises an RNA export signal. Exemplary RNA exportsequences include but are not limited to sequences from woodchuckhepatitis virus post-transcriptional element (WPRE). The woodchuckhepatitis virus (WHV) post-transcriptional regulatory element (Wpre)significantly increases transgene expression in target cells, byincreasing RNA stability in a transgene, promoter and vector-independentmanner (Zuffrey et al, 1999). However, it can express a truncated60-amino acid protein derived from the WHV X gene involved in livercancer (Kingsman et al, 2005). Therefore, most pre-clinical protocolsand clinical trials include a mutated version of the Wpre element(Zanta-Boussif et al, 2009). On the other hand, the use of two SV40-USEelements in SIN-LV vectors has been seen to be more efficient than WPREsequence in suppressing transcriptional read through (Schambach et al,2007). More precisely, the WPRE disclosed herein is a chimeric WPRE thatcarries 589 nucleotides from the modified WPRE performed by AxelSchambach (nucleotides 1-589) (WO 2008136670 A2; [5]) and 88 from aformer WPRE (nucleotide 590-677) (Zuffrey et al, 1999). Data disclosedherein shows this chimeric wpre works better than the former WPRE. Thechimeric WPRE sequence comprises the sequence listed in the table below.

TABLE 1 Modified WPRE sequenceCGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAGGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO: 1)

The present invention also include a nucleic acid, e.g., apolynucleotide sequence, comprising a sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identity to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:9. Inparticular embodiments, the polynucleotide sequence comprises thesequence set forth in SEQ ID NO:1 or SEQ ID NO:9.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the Wpre sequence comprises orconsists of the sequence of SEQ ID NO:1, or a sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% identity to the sequence of SEQ ID NO:1.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the Wpre sequence comprises orconsists of the following sequence, or a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identity to the following sequence:

(SEQ ID NO: 9) CGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAGGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG.

In certain embodiments, an expression cassette or gene delivery vector,e.g., a lentivirus, comprises a polynucleotide sequence comprising thefollowing sequences in 5′ to 3′ order:

(a) a PGK promoter sequence, optionally a human PGK promoter sequence;

(b) a sequence encoding a pyruvate kinase polypeptide, optionally acodon optimized RPK coding or cDNA sequence; and

(c) a mutant Wpre sequence, optionally comprising or consisting of thesequence of SEQ ID NO:1.

In certain embodiments, an expression cassette or gene delivery vector,e.g., a lentivurus, comprises a polynucleotide sequence comprising thefollowing sequences in 5′ to 3′ order:

(a) a cPPT sequence;

(b) PGK promoter sequence, optionally a human PGK promoter sequence;

(c) a sequence encoding a pyruvate kinase polypeptide, optionally acodon optimized RPK coding or cDNA sequence; and

(d) a mutant Wpre sequence, optionally comprising or consisting of thesequence of SEQ ID NO:1.

In certain embodiments, an expression cassette or gene delivery vector,e.g., a lentivurus, comprises a polynucleotide sequence comprising thefollowing sequences in 5′ to 3′ order:

(a) a 5′ LTR, optionally a modified 5′ LTR;

(b) a cPPT sequence;

(c) PGK promoter sequence, optionally a human PGK promoter sequence;

(d) a sequence encoding a pyruvate kinase polypeptide, optionally acodon optimized RPK coding or cDNA sequence;

(e) a mutant Wpre sequence, optionally comprising or consisting of thesequence of SEQ ID NO:1; and

(f) a 3′ LTR, optionally a modified 3′ LTR.

In certain embodiments, the gene delivery vector is PGK-coRPK LV, orcomprises the elements depicted in FIG. 21.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the codon optimized RPK cDNA orcoding sequence encodes a PKLR polypeptide that comprises or consists ofthe sequence disclosed in any of GenBank accession Nos. XP_016856982.1,XP)_011507942.1, XP_006711449.1, NP_870986.1, or NP_000289.1, or afunctional fragment of any of these sequences, or a sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to any of these sequences.

Other combinations of elements both as disclosed herein or as known inthe art will be readily appreciated by the ordinarily skilled artisan.

Additionally, as will be recognized by one of ordinary skill in the art,the polynucleotide cassettes may optionally contain other elementsincluding, but not limited to restriction sites to facilitate cloningand regulatory elements for a particular gene expression vector.

In some aspects of the present invention, the subject polynucleotidecassettes are used to deliver a gene to cells of an animal, e.g. todetermine the effect that the gene has on cell viability and/orfunction, to treat a cell disorder, etc. Accordingly, in some aspects ofthe invention, the composition that provides for the expression of atransgene in mammalian cells is a gene delivery vector, wherein the genedelivery vector comprises the polynucleotide cassettes of the presentdisclosure.

Any convenient gene therapy vector that finds use deliveringpolynucleotide sequences to mammalian cells is encompassed by the genedelivery vectors of the present disclosure. For example, the vector maycomprise single or double stranded nucleic acid, e.g. single stranded ordouble stranded DNA. For example, the gene delivery vector may be DNA,e.g., a naked DNA, e.g. a plasmid, a minicircle, etc. The vector maycomprise single-stranded or double-stranded RNA, including modifiedforms of RNA. In another example, the gene delivery vector may be anRNA, e.g., an mRNA or modified mRNA.

As another example, the gene delivery vector may be a viral vectorderived from a virus, e.g. an adenovirus, an adeno-associated virus, alentivirus (LV), a herpes virus, an alpha virus or a retrovirus, e.g.,Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus(MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus(FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus(MSCV) and Rous Sarcoma Virus (RSV)) or lentivirus. While embodimentsencompassing the use of lentivirus are described in greater detailbelow, it is expected that the ordinarily skilled artisan willappreciate that similar knowledge and skill in the art can be brought tobear on non-LV gene therapy vectors as well. In some embodiments, thegene delivery vector is a self-limiting lentivirus.

In such embodiments, the subject polynucleotide cassette is flanked onthe 5′ and 3′ ends by functional long terminal repeat (LTR) sequences.In one embodiment, the position of different elements present in thebackbone of the lentiviral vector is depicted in FIG. 1. Both LTRsequences have been modified to generate self-inactivating (SIN) LVvectors. SIN vectors have a 400 by deletion in the 3′-LTR, covering thepromoter/enhancer elements from the U3 region. Expression of thetransgene is thereby dependent on internal promoters, reducing the riskof RCLs and decreasing promoter interference (Ginn et al, 2003). This3′LTR deletion removes the TATA box, preventing transcription initiation(Miyoshi et al. 1998; Zuffrey et al 1998); and therefore inactivatingthe vector. The U3 region of the 5′-LTR has been replaced by otherheterologous promoting sequences (i.e. CMV or RSV) to achieve aTat-independent transcription and to increase genomic RNA synthesis,resulting in the increase of the viral titer. Because 5′-U3 regiondrives the expression of primary transcripts, its modifications will notbe present in transduced cells (Schambach et al. 2009). Exogenouselements, such as β-globin or SV40 polyadenylation signals (Iwakuma etal, 1999) or the upstream sequence element (USE) from simian virus 40(SV40-USE) (Schambach et al. 2007), have also been included in the Rregion of the viral 3′LTR in order to decrease the transcriptionalreadthrough from the internal promoters (Zaiss et al, 2002) or fromremnants of the deleted U3 region of SIN-LV vectors (Almarza et al.2011) preventing the potential transcriptional activation of thedownstream genes. The leader region contains the packaging signal (Ψ),and LV vectors were thought to require approximately 300 by of the Gaggene in this region. Currently, this Gag sequence has been reduced tojust 40 by (FIG. 1). The Rev responsive element (RRE) has also beenincluded to improve the efficiency of gene transfer, although itcontains surrounding Env remnants. The central polypurine tract (cPPT),which facilitates nuclear translocation of the pre-integrationcomplexes, together with the central terminal sequence (CTS) involved inthe separation of reverse transcriptase, has been seen to improve viraltiter (Zennou, et al. 2000; Follenzi et al. 2000). In particularembodiments, the cPPT present in any of the expression cassettes or genedelivery vectors described herein comprises or consists of the followingsequence: TTTAAAAGAAAAGGGGGGATTGGGGGGT (SEQ ID NO:2), or a sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% identity to SEQ ID NO:2.

The dNEF/PPT signal is essential for reverse transcription, and itsincorporation significantly improves LV vector production.

Gene therapy vectors encapsulating the polynucleotide cassettes of thepresent disclosure may be produced using standard methodology. Forexample, in the case of LV virions, an LV expression vector according tothe invention may be introduced into a producer cell, followed byintroduction of an LV helper construct, where the helper constructincludes LV coding regions capable of being expressed in the producercell and which complement LV helper functions absent in the LV vector.This is followed by introduction of helper virus and/or additionalvectors into the producer cell, wherein the helper virus and/oradditional vectors provide accessory functions capable of supportingefficient LV virus production. The producer cells are then cultured toproduce LV. These steps are carried out using standard methodology.

Any suitable method for producing viral particles for delivery of thesubject polynucleotide cassettes can be used, including but not limitedto those described in the examples that follow. Any concentration ofviral particles suitable to effectively transducer mammalian cells canbe prepared for contacting mammalian cells in vitro or in vivo. Forexample, the viral particles may be formulated at a concentration of 10⁸vector genomes per ml or more, for example, 5×10⁸ vector genomes per mL;10⁹ vector genomes per mL; 5×10⁹ vector genomes per mL, 10¹⁰ vectorgenomes per mL, 5×10¹⁹ vector genomes per mL; 10¹¹ vector genomes permL; 5×10¹¹ vector genomes per mL; 10¹² vector genomes per mL; 5×10¹²vector genomes per mL; 10¹³ vector genomes per mL; 1.5×10¹³ vectorgenomes per mL; 3×10¹³ vector genomes per mL; 5×10¹³ vector genomes permL; 7.5×10¹³ vector genomes per mL; 9×10¹³ vector genomes per mL; 1×10¹⁴vector genomes per mL, 5×10¹⁴ vector genomes per mL or more, buttypically not more than 1×10¹⁵ vector genomes per mL.

In preparing the subject LV compositions, any host cells for producingLV virions may be employed, including, for example, mammalian cells(e.g. 293 cells), insect cells (e.g. SF9 cells), microorganisms andyeast. Host cells can also be packaging cells in which the LV rep andcap genes are stably maintained in the host cell or producer cells inwhich the LV vector genome is stably maintained and packaged. Exemplarypackaging and producer cells are derived from SF-9, 293, A549 or HeLacells. LV vectors are purified and formulated using standard techniquesknown in the art.

In certain embodiments, the present invention includes a cell comprisingan expression cassette or gene delivery vector disclosed herein. Inrelated embodiments, the cell is transduced with a viral vectorcomprising an expression cassette disclosed herein or has an expressioncassette disclosed herein integrated into the cell's genome. In certainembodiments, the cell is a cell used to produce a viral gene deliveryvector. In other embodiments, the cell is a cell to be delivered to asubject in order to provide to the subject the gene product encoded bythe expression cassette. Thus, in certain embodiments, the cell isautologous to the subject to be treated or was obtained from the subjectto be treated. In other embodiments, the cell is allogeneic to thesubject to be treated or was obtained from a donor other than thesubject to be treated. In particular embodiments, the cell is amammalian cell, e.g., a human cell. In certain embodiments, the cell isa blood cell, an erythrocyte, a hematopoietic progenitor cell, a bonemarrow cell, e.g., a lineage depleted bone marrow cell, a hematopoieticstem cell (e.g., CD34+) or a committed hematopoietic erythroidprogenitor cell

The present invention includes pharmaceutical compositions comprising apolynucleotide cassette, gene delivery vector, or cell described hereinand a pharmaceutically-acceptable carrier, diluent or excipient. Thesubject polynucleotide cassette, gene delivery vector, or cell can becombined with pharmaceutically-acceptable carriers, diluents andreagents useful in preparing a formulation that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor primate use. Such excipients can be solid, liquid, semisolid, or, inthe case of an aerosol composition, gaseous. Examples of suchexcipients, carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. Supplementary active compounds can also be incorporated intothe formulations. Solutions or suspensions used for the formulations caninclude a sterile diluent such as water for injection, saline solution,fixed oils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial compounds such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating compounds such as ethylenediaminetetraacetic acid (EDTA);buffers such as acetates, citrates or phosphates; detergents such asTween 20 to prevent aggregation; and compounds for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Inparticular embodiments, the pharmaceutical compositions are sterile.

Pharmaceutical compositions suitable for use in the present inventionfurther include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion.

Sterile solutions can be prepared by incorporating the active compoundin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In one embodiment, the compositions are prepared with carriers that willprotect the gene cassette or expression vector against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially.

It is especially advantageous to formulate oral, ocular or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser, e.g. syringe, e.g. a prefilled syringe, together withinstructions for administration.

The pharmaceutical compositions of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal comprising ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof.

The term “pharmaceutically acceptable salt” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Avariety of pharmaceutically acceptable salts are known in the art anddescribed, e.g., in in “Remington's Pharmaceutical Sciences”, 17thedition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa.,USA, 1985 (and more recent editions thereof), in the “Encyclopaedia ofPharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), InformaHealthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2(1977). Also, for a review on suitable salts, see Handbook ofPharmaceutical Salts: Properties, Selection, and Use by Stahl andWermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Metals used as cations comprise sodium, potassium, magnesium, calcium,and the like. Amines comprise N—N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119).The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentinvention.

The subject polynucleotide cassette, gene delivery vector, e.g.,recombinant virus (virions), or cell (e.g., transduced with a genedelivery vector disclosed herein) can be incorporated intopharmaceutical compositions for administration to mammalian patients,particularly primates and more particularly humans. The subjectpolynucleotide cassette, gene delivery vector, e.g. virions, or cell canbe formulated in nontoxic, inert, pharmaceutically acceptable aqueouscarriers, preferably at a pH ranging from 3 to 8, more preferablyranging from 6 to 8. Such sterile compositions will comprise the vectoror virion containing the nucleic acid encoding the therapeutic moleculedissolved in an aqueous buffer having an acceptable pH uponreconstitution.

In some embodiments, the pharmaceutical composition provided hereincomprise a therapeutically effective amount of a cell, vector or viriondisclosed herein in admixture with a pharmaceutically acceptable carrierand/or excipient, for example saline, phosphate buffered saline,phosphate and amino acids, polymers, polyols, sugar, buffers,preservatives and other proteins. Exemplary amino acids, polymers andsugars and the like are octylphenoxy polyethoxy ethanol compounds,polyethylene glycol monostearate compounds, polyoxyethylene sorbitanfatty acid esters, sucrose, fructose, dextrose, maltose, glucose,mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose,trehalose, bovine or human serum albumin, citrate, acetate, Ringer's andHank's solutions, cysteine, arginine, carnitine, alanine, glycine,lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycol.Preferably, this formulation is stable for at least six months at 4° C.

In some embodiments, the pharmaceutical composition provided hereincomprises a buffer, such as phosphate buffered saline (PBS) or sodiumphosphate/sodium sulfate, tris buffer, glycine buffer, sterile water andother buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. (1966) Biochemistry 5:467. The pH of the bufferin which the pharmaceutical composition comprising the tumor suppressorgene contained in the adenoviral vector delivery system, may be in therange of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to7.4.

In certain embodiments, viral vectors may be formulated into anysuitable unit dosage, including, without limitation, 1×10⁸ vectorgenomes or more, for example, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³vector genomes or more, in certain instances, 1×10¹⁴ vector genomes, butusually no more than 4×10¹⁵ vector genomes. In some cases, the unitdosage is at most about 5×10¹⁵ vector genomes, e.g. 1×10¹⁴ vectorgenomes or less, for example 1×10¹³, 1×10¹², 1×10¹¹, 1×10¹⁰, or 1×10⁹vector genomes or less, in certain instances 1×10⁸ vector genomes orless, and typically no less than 1×10⁸ vector genomes. In some cases,the unit dosage is 1×10¹ to 1×10¹¹ vector genomes. In some cases, theunit dosage is 1×10¹⁰ to 3×10¹² vector genomes. In some cases, the unitdosage is 1×10⁹ to 3×10¹³ vector genomes. In some cases, the unit dosageis 1×10⁸ to 3×10¹⁴ vector genomes. In one embodiment, the range is fromabout 5×10¹⁰ to about 1×10¹¹ vector genomes. In some embodiments, therange is from about 1×10⁹ to about 1×10¹⁰ vector genomes.

In some cases, the unit dosage of a pharmaceutical composition may bemeasured using multiplicity of infection (MOI). By MOI it is meant theratio, or multiple, of vector or viral genomes to the cells to which thenucleic acid may be delivered. In some cases, the MOI may be 1×10⁶. Insome cases, the MOI may be 1×10⁵-1×10⁷. In some cases, the MOI may be1×10⁴-1×10⁸. In some cases, recombinant viruses of this disclosure areat least about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷,and 1×10¹⁸ MOI. In some cases, recombinant viruses of this disclosureare 1×10⁸ to 3×10¹⁴ MOI_In some cases, recombinant viruses of thedisclosure are at most about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶,1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵,1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In some, embodiments the range is fromabout 20 to about 400 MOI.

In some aspects, the amount of pharmaceutical composition comprisesabout 1×10⁸ to about 1×10¹⁵ recombinant viruses, about 1×10⁹ to about1×10¹⁴ recombinant viruses, about 1×10¹⁰ to about 1×10¹³ recombinantviruses, or about 1×10¹¹ to about 3×10¹² recombinant viruses.

Methods

As disclosed herein, the subject polynucleotide cassettes and genedelivery vectors, referred to collectively herein as the “subjectcompositions”, find use in expressing a transgene in cells of an animal.For example, the subject compositions may be used in research, e.g. todetermine the effect that the gene has on cell viability and/orfunction. As another example, the subject compositions may be used inmedicine, e.g. to treat or prevent a disease or disorder. Thus, in someaspects of the invention, methods are provided for the expression of agene in cells, the method comprising contacting cells with a compositionof the present disclosure. In some embodiments, contacting occurs invitro or ex vivo. In some embodiments, contacting occurs in vivo, i.e.,the subject composition is administered to a subject.

For instances in which mammalian cells are to be contacted in vitro orex vivo with a subject polynucleotide cassette or gene delivery vectorcomprising a subject polynucleotide cassette, the cells may be from anymammalian species, e.g. rodent (e.g. mice, rats, gerbils, squirrels),rabbit, feline, canine, goat, ovine, pig, equine, bovine, primate,human. Cells may be from established cell lines or they may be primarycells, where “primary cells”, “primary cell lines”, and “primarycultures” are used interchangeably herein to refer to cells and cellscultures that have been derived from a subject and allowed to grow invitro for a limited number of passages, i.e. splittings, of the culture.For example, primary cultures are cultures that may have been passaged 0times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but notenough times go through the crisis stage. Typically, the primary celllines of the present invention are maintained for fewer than 10 passagesin vitro.

Embodiments of the present invention comprise mammalian cells (e.g.,CD34+ cells) transduced with a viral delivery vector, e.g., a lentiviralvector containing the human liver and erythroid pyruvate kinase (PKLR)gene. Accordingly, the present invention includes a method oftransducing a mammalian cell, e.g. a human hematopoietic stem cell orother cell described herein, comprising contacting the cell with a viraldelivery vector, e.g., a lentiviral vector, comprising an expressioncassette described herein. In certain embodiments, the cell waspreviously obtained from a subject to be treated, or from another donor.In particular embodiments, the subject was diagnosed with PKD, and thecell is transduced with a LV comprising an expression cassette encodingpyruvate kinase, e.g., a codon-optimized RPK coding region or cDNA. Itis understood that the disclosed methods, e.g., those used to deliver apyruvate kinase gene product, e.g., using a coPRK cDNA sequence, to asubject may also be used to treat hemolytic anemia, and/or normalizeerythroid differentiation, increase the number of functional matureerythrocytes, reduce extramedullar erythropoiesis, reduce splenomegalyand other secondary effects of hemolytic anemia or PKD.

To promote expression of the transgene, the subject polynucleotidecassette or gene delivery vector comprising a subject polynucleotidecassette will be contacted with the cells for about 30 minutes to 24hours or more, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours,18 hours, 20 hours, 24 hours, etc.

The subject polynucleotide cassette or gene delivery vector comprising asubject polynucleotide cassette may be provided to the subject cells oneor more times, e.g. one time, twice, three times, or more than threetimes, and the cells allowed to incubate with the agent(s) for someamount of time following each contacting event e.g. 16-24 hours, afterwhich time the media is replaced with fresh media and the cells arecultured further. Contacting the cells may occur in any culture mediaand under any culture conditions that promote the survival of the cells.The culture may contain growth factors to which the cells areresponsive. Growth factors, as defined herein, are molecules capable ofpromoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors.

Typically, an effective amount of subject polynucleotide cassette orgene delivery vector comprising a subject polynucleotide cassette isprovided to produce the expression of the transgene in cells. Asdiscussed elsewhere herein, the effective amount may be readilydetermined empirically, e.g. by detecting the presence or levels oftransgene gene product, by detecting an effect on the viability orfunction of the cells, etc. Typically, an effect amount of subjectpolynucleotide cassette or gene delivery vector comprising a subjectpolynucleotide cassette will promote greater expression of the transgenein cells than the same amount of a polynucleotide cassette as known inthe art. Typically, expression will be enhanced 2-fold or more relativeto the expression from a reference, or control, polynucleotide cassettee.g. as known in the art, for example 3-fold, 4-fold, or 5-fold or more,in some instances 10-fold, 20-fold or 50-fold or more, e.g. 100-fold.

For instances in which cells are to be contacted in vivo with a subjectpolynucleotide cassette or gene delivery vector comprising a subjectpolynucleotide cassette, the subject may be any mammal, e.g. rodent(e.g. mice, rats, gerbils), rabbit, feline, canine, goat, ovine, pig,equine, bovine, or primate. In a further preferred embodiment, theprimate is a human. In a further embodiment, the cells are CD34+ cells.

The methods and compositions of the present disclosure find use, e.g.,in the treatment of pyruvate kinase deficiency.

In another embodiment, the present invention includes a method oftreating a disease in a subject in need thereof comprising providing tothe subject an effective amount of cells transduced with a gene deliveryvector, e.g., a viral vector, that expresses a therapeutic gene productin the cells. In particular embodiments, the cells are autologous to thesubject. In certain embodiments, the cells are erythroid cells, e.g.,hematopoietic stem cells or committed hematopoietic erythroid progenitorcells. In some embodiments, the cell is a bone marrow cell, e.g., alineage depleted bone marrow cell. In particular embodiments, the methodis used to treat PKD, and the viral vector is a LV comprising anexpression construct disclosed herein comprising a human PGK promoteroperably linked to a codon optimized human PKLR gene cDNA or codingsequence, and a mutated Wpre disclosed herein. In particularembodiments, the cells are provided to the subject parenterally, e.g.,via intravenous injection.

In another embodiment, the present invention includes a method oftreating PKD in a subject in need thereof, comprising providing to thesubject an effective amount of autologous C34+ stem cells transducedwith a lentiviral vector that expresses a codon optimized PKLR cDNA inthe cells, wherein the lentiviral vector comprises a human PGK promoteroperably linked to the codon optimized human PKLR cDNA or codingsequence, and a mutated Wpre sequence disclosed herein. In particularembodiments, the cells are hematopoietic stem cells or committedhematopoietic erythroid progenitor cells, e.g., bone marrow cells. Inparticular embodiments, the cells are provided to the subjectparenterally, e.g., via intravenous injection.

In another embodiment, the present invention provides a of treating adisease in a subject in need thereof comprising providing to the subjectan effective amount of a gene delivery vector, e.g., a viral vector,that expresses a therapeutic gene product in the subject. In particularembodiments, the method is used to treat PKD, and the viral vector is aLV comprising an expression construct disclosed herein comprising ahuman PGK promoter operably linked to a codon optimized human PKLR genecDNA or coding sequence, and a mutated Wpre disclosed herein. Inparticular embodiments, the gene delivery vector are provided to thesubject parenterally, e.g., via intravenous injection.

In particular embodiments, the cells or gene delivery vectors areprovided to the subject in pharmaceutical compositions.

In some embodiments, the subject methods result in a therapeuticbenefit, e.g. preventing the development of a disorder, halting theprogression of a disorder, reversing the progression of a disorder, etc.In some embodiments, the subject method comprises the step of detectingthat a therapeutic benefit has been achieved. The ordinarily skilledartisan will appreciate that such measures of therapeutic efficacy willbe applicable to the particular disease being modified, and willrecognize the appropriate detection methods to use to measuretherapeutic efficacy.

Expression of the transgene using the subject transgene is expected tobe robust. Accordingly, in some instances, the expression of thetransgene, e.g. as detected by measuring levels of gene product, bymeasuring therapeutic efficacy, etc. may be observed two months or lessafter administration, e.g. 4, 3 or 2 weeks or less after administration,for example, 1 week after administration of the subject composition.Expression of the transgene is also expected to persist over time.Accordingly, in some instances, the expression of the transgene, e.g. asdetected by measuring levels of gene product, by measuring therapeuticefficacy, etc., may be observed 2 months or more after administration ofthe subject composition, e.g., 4, 6, 8, or 10 months or more, in someinstances 1 year or more, for example 2, 3, 4, or 5 years, in certaininstances, more than 5 years.

In certain embodiments, the method comprises the step of detectingexpression of the transgene in the cells or in the subject, whereinexpression is enhanced relative to expression from a polynucleotidecassette not comprising the one or more improved elements of the presentdisclosure. Typically, expression will be enhanced 2-fold or morerelative to the expression from a reference, i.e. a controlpolynucleotide cassette, e.g. as known in the art, for example 3-fold,4-fold, or 5-fold or more, in some instances 10-fold, 20-fold or 50-foldor more, e.g. 100-fold, as evidenced by, e.g. earlier detection, higherlevels of gene product, a stronger functional impact on the cells, etc.

Typically, if the subject composition is an LV comprising the subject apolynucleotide cassette of the present disclosure, an effective amountto achieve a change in will be about 1×10⁸ vector genomes or more, insome cases 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³ vector genomes ormore, in certain instances, 1×10¹⁴ vector genomes or more, and usuallyno more than 1×10¹⁵ vector genomes. In some cases, the amount of vectorgenomes that is delivered is at most about 1×10¹⁵ vector genomes, e.g.1×10¹⁴ vector genomes or less, for example 1×10¹³, 1×10¹², 1×10¹¹,1×10¹⁰, or 1×10⁹ vector genomes or less, in certain instances 1×10⁸vector genomes, and typically no less than 1×10⁸ vector genomes. In somecases, the amount of vector genomes that is delivered is 1×10¹⁰ to1×10¹¹ vector genomes. In some cases, the amount of vector genomes thatis delivered is 1×10¹⁰ to 3×10¹² vector genomes. In some cases, theamount of vector genomes that is delivered is 1×10⁹ to 3×10¹³ vectorgenomes. In some cases, the amount of vector genomes that is deliveredis 1×10⁸ to 3×10¹⁴ vector genomes.

In some cases, the amount of pharmaceutical composition to beadministered may be measured using multiplicity of infection (MOD. Insome cases, MOI may refer to the ratio, or multiple of vector or viralgenomes to the cells to which the nucleic may be delivered. In somecases, the MOI may be 1×10⁶. In some cases, the MOI may be 1×10⁵-1×10⁷.In some cases, the MOI may be 1×10⁴-1×10⁸. In some cases, recombinantviruses of the disclosure are at least about 1×10¹, 1×10², 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In some cases,recombinant viruses of this disclosure are 1×10⁸ to 3×10¹⁴ MOI. In somecases, recombinant viruses of the disclosure are at most about 1×10¹,1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI.

In some aspects, the amount of pharmaceutical composition comprisesabout 1×108 to about 1×10¹⁵ particles of recombinant viruses, about1×10⁹ to about 1×10¹⁴ particles of recombinant viruses, about 1×10¹⁰ toabout 1×10¹³ particles of recombinant viruses, or about 1×10¹¹ to about3×10¹² particles of recombinant viruses.

Any total number of viral particles suitable to provide appropriatetransduction of cells to confer the desired effect or treat the diseasecan be administered to the mammal. In various preferred embodiments, atleast 10⁸; 5×10⁸; 10⁹; 5×10⁹, 10¹⁰, 5×10¹⁰, 10¹¹, 5×10¹¹; 10¹²; 5×10¹²;10¹³; 10¹⁴ viral particles or more, but typically not more than 1×10¹⁵viral particles are injected. Any suitable number of administrations ofthe vector to the mammal or the primate eye can be made. In oneembodiment, the methods comprise a single administration; in otherembodiments, multiple administrations are made over time as deemedappropriate by an attending clinician. In some embodiments at least2×10⁸ VG/ml of 5×10⁵ cells/ml is required in a single administration (24hours transduction) to result in high transduction efficiencies.

Individual doses are typically not less than an amount required toproduce a measurable effect on the subject, and may be determined basedon the pharmacokinetics and pharmacology for absorption, distribution,metabolism, and excretion (“ADME”) of the subject composition or itsby-products, and thus based on the disposition of the composition withinthe subject. This includes consideration of the route of administrationas well as dosage amount. Effective amounts of dose and/or dose regimencan readily be determined empirically from preclinical assays, fromsafety and escalation and dose range trials, individualclinician-patient relationships, as well as in vitro and in vivo assayssuch as those described herein and illustrated in the Examples.

Several aspects of the invention are described herein with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events. Furthermore, not allillustrated acts or events are required to implement a methodology inaccordance with the present invention.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application.Nothing-herein is to be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention. Further, the dates of publication provided may bedifferent from the actual publication dates which may need to beindependently confirmed.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety, e.g., to disclose and describe the methodsand/or materials in connection with which the publications are cited. Itis understood that the present disclosure supersedes any disclosure ofan incorporated publication to the extent there is a contradiction.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Experimental Methods

Vectors and lentiviral supernatant production. LVs were generated asdescribed herein. CoRPK sequence was designed using the GeneArt®software to increase the GC content of the sequence and to preventcryptic splice sites. Vectors were developed using thepCCL.sin.ppt.hPGK-EGFP-Wpre* construct as backbone, generously providedby Dr. Naldini (HSR-TIGET, San Raffaele Telethon Institute, Milano,Italy). Vector stocks of VSVG pseudotyped LVs were prepared by 3-plasmidcalcium phosphate-mediated transfection in 293T cells (ATCC: CRL-1573,Rockeville, Md., USA), as previously described [Follenzi A, et al.(2000). Nat Genet 25: 217-222]. Titers of infective LVs were determinedin HT1080 cells (ATCC: CCL-121) by qPCR as described elsewhere [CharrierS, et al. (2005). Gene Ther 12: 597-606]. Lentiviral stocks of 10⁷-10⁸viral particles (vp)/mL titers were routinely obtained.

Purification and transduction of murine HSCs. BM from 8-14 week-old malePKD mice was harvested from the leg bones and lineage negative cells(Lin⁻) were purified using the Lin⁻ Cell Depletion kit (Miltenyi Biotec,Gladbach, Germany), obtaining 70-90% purity. Lin⁻ cells werepre-stimulated with 100 ng/mL of recombinant human IL-11 (Peprotech ECLtd., London, UK) and 100 ng/mL of recombinant murine SCF (R&D SystemsInc., Minneapolis, Minn.) in IMDM-Glutamax medium supplemented with 20%FBS and 0.5% antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin,(Thermo Fisher Scientific, Waltham, Mass.) for 24 h, and then transducedwith EGFP or coRPK carrying LVs in two cycles of transduction at MOIs of1-10 vp/cell. Each transduction was carried out for 24 h in the presenceof the aforementioned cytokines on plates previously coated with CH-296fibronectin fragment (2 μg/cm²; Retronectin, TakaraShuzo, Otsu, Japan)overnight at 4° C.

In vivo RBC survival. Transplanted mice carrying the coRPK transgenewere injected with three consecutive intravenous injections (12 h apart)of Biotin 3-sulfo-N-hydroxysuccinimide ester sodium salt (50 mg/kg)(Sigma Aldrich, Saint Louis, Mo.). Twelve hours after the lastinjection, tail vein blood was harvested and labelled with 2 μg/mL ofanti-mouse Ter119-PE (BD Bioscience, San Jose, Calif.) andstreptavidin-FITC (50 μg/mL, BD Biosciences, San Jose, Calif.) for 30min at 4° C. Samples were analyzed in an EPICS XL flow cytometer(Beckman Coulter, Brea, Calif.) every 2-4 days for 40 days after theinjection. RBC survival kinetics was measured by the percentage ofbiotinylated cells within the total RBC population.

CFC Assay. CFC assay was performed in BM and spleen from control andtransplanted mice according to manufacturer's procedure from Methocultmedium GF M3434 (Stem Cell Technologies, Vancouver, Canada). BM cellswere harvested at different time-points after transplant from all groupsof mice, and CFUs (clusters of 30 or more cells) were scored 7 daysafter seeding in a Nikon Diaphot-TMD microscope.

Identification of hematopoietic lineages. PBMCs were obtained from thetail vein of transplanted animals and labelled with a panel ofantibodies to detect different hematopoietic cells. Myeloid cells weredetected with anti-GR-1 and anti-Mac-1 biotinylated antibodies (BDBioscience, San Jose, Calif., 5 μg/mL), while lymphoid cells weredetected using anti-CD3-PE antibody for T-cells, and anti-B220-PE andanti-B220-PECyS antibodies for B-cells (BD Bioscience, San Jose, Calif.,10 μg/mL), together with SAV-TRC secondary antibody (Invitrogen, ThermoFisher Scientific, Waltham, Mass.). Samples were analysed in a BD LSRFortessa Cytometer (BD Bioscience, San Jose, Calif.″ USA) adding DAPI(Boehringer, Ingelheim, Germany, 2 μg/mL) to exclude death cells.

Structural and histological studies. Spleens were collected,photographed and weighed on precision scales to determine the presenceof splenomegaly. Histological studies were performed on spleen and liversections obtained following conventional histological methods, andstained with hematoxylin (Gill-2 Haematoxylin, Thermo, Pittsburgh, USA)and eosin (Eosin Alcoholic, Thermo Fisher Scientific, Waltham, Mass.).Iron deposits were also studied in the spleen by Prussian Blue or Perls'staining (Sigma Aldrich, Saint Louis, Mo.) following manufacturer'sinstructions. All sections were examined using an Olympus BX40 lightmicroscope and photographed with an Olympus DP21 camera, with a finalmagnification of 100× or 200×.

Erythroid differentiation. Flow cytometry analysis of Ter119 and CD71marker intensities in BM and spleen were used to identify the differenterythroid subpopulations as described elsewhere [Socolovsky M, et al.(2001). Blood 98: 3261-3273] using 4 μg/mL of anti-mouse Ten 19-PEantibody (BD Bioscience, San Jose, Calif.), 10 μg/mL of biotinylatedanti-CD71 antibody (BD Bioscience, San Jose, Calif.) andstreptavidin-tricolor (Invitrogen, Thermo Fisher Scientific, Waltham,Mass.). Cells were then analyzed in an EPICS XL flow cytometer (BeckmanCoulter, Brea, Calif.) using propidium iodide (IP, 2 μg/mL) to detectlive cells.

Provirus quantification. Detection and quantification of integratedprovirus per cell was accomplished using complementary primers to thepackaging proviral sequence (Ψ) and the mouse Titin housekeeping gene.Total BM and peripheral blood samples were collected periodically, andgenomic DNA from nucleated cells was isolated using the DNeasy Blood &Tissue kit (Qiagen, Venlo, Limburg, The Netherlands). Twenty to 50 ng ofgenomic DNA (gDNA) were amplified by multiplex qPCR using the 7500 FastReal-Time PCR System (Applied Biosystems, Thermo Fisher Scientific,Waltham, Mass.) and primers and probes previously described [Charrier S,et al. (2011). Gene Ther 18: 479-487].

Chimerism. Presence of donor cells was quantified by qPCR detecting theY chromosome SRY gene and the mouse β-Actin housekeeping gene. Primersand probes previously described [Navarro S et al (2006). Mol Ther 14:525-535] were used and genomic DNA from PB of transplanted mice wasamplified using the 7500 Fast Real-Time PCR System (Applied Biosystems,Thermo Fisher Scientific, Waltham, Mass.). Standard curves were preparedusing gDNA extracts from samples containing 0% to 100% of BM cells frommale/female mouse mixtures and chimerism was calculated as: % of donorengraftment=100×2^((CtβAct-CtSRY)).

LAM-PCR procedure. In order to identify vector integration sites, 3′vector LTRgenome junctions were amplified by LAM-PCR following themethod published by Schmidt et al. 2007 [Nat Methods 4: 1051-1057]. Thestarting linear amplification (100 cycles) was performed usingbiotinylated LTR specific primers and up to 100 ng of gDNA as template.Linear amplification products were purified using streptavidin magneticbeads and followed by complementary strand synthesis, parallel digestionwith 2 different restriction enzymes (Tsp509I and HpyCH4IV) and twoligation reactions using linker cassettes complementary to the ends leftby the enzyme's cut. The fragments generated were amplified by twoadditional exponential PCR steps. LAM-PCR products were separated andquantified by gel electrophoresis on a MultiNA automated system(Shimadzu).

Setup of LAM-PCR products for Illumina MiSeq sequencing. Following themethod published by Parazynski et al [Paruzynski A, et al. (2010). NatProtoc 5: 1379-1395], 40 ng of the second exponential PCR productsgenerated by Tsp5091 and HpyCH4IV enzymes were re-amplified using fusionprimers containing specific sequences that allow paired end sequencingon an Illumina MiSeq sequencer. LAM-PCR samples were adapted for454-pyrusequencing by fusion PCR to add the Roche 454 GS-FLX adaptors:adaptor A plus an 8-nucleotide barcode was added to the LTR end of theLAM-PCR amplicon; adaptor B was added to the linker cassette side. In5′-3′ orientation the final amplicon was composed as follow: adaptor A,barcode, LTR sequence, unknown genome sequence, linker cassette sequenceand primer B. Purified fusion primer PCR products were run andquantified on a MultiNA automated electrophoresis system, and pooledtogether in order to obtain a final equimolar library of 10 nM. Thefinal library was then re-quantified using a KAPA Library QuantificationKit for Illumina Sequencing Platform (Kapa Biosystems, Wilmington,Mass.) on a Viia7 real-time PCR system (Applied Biosystems, ThermoFisher Scientific, Waltham, Mass.), obtaining an estimated concentrationof 16.35 nM. Finally, libraries were sequenced using the Illumina MiSeqReagent Kit.

Bioinformatics analysis. To extract vector integration sites (IS) from ahigh-throughput sequencing platform, both Roche 454 and IlluminaMiSeq/HiSeq, a pipeline taking in input the row data (typically in FastQfile format) was designed, providing the list of reliable IS and thenearest gene. Superior level analyses for clonal abundancequantification and gene ontology enrichment were performed using Excel,GraphPad Prism™ and available online tools.

NGS data processing and pipeline usage. The step of NGS data processingdeals with the management of high-throughput data from Illumina MiSeqsequencing platforms and aims at identify IS in which all valid sequencereads are aligned to the reference genome. Data processing comprises twomain activities: 1. Data quality inspection and analysis, in whichlentiviral vector sequences and other contaminants are trimmed. 2.Integration site identification, in which all valid sequence reads arealigned to the genome of reference and valid ISs are retrieved.

Data quality analysis. In order to identify IS from Illumina MiSeq rawdata a bioinformatics pipeline was developed. Standard LAM-PCR productscontain a LTR sequence, a flanking human genomic sequence and a linkercassette (LC) sequence. The 459 technology allowed retrieval of LAM-PCRsequences with length ranging from 10 bp to 900 bp. Similar results wereretrieved from Illumina MiSeq paired-ends reads. These length boundariesare important parameters to consider in the quality analysis processsince they affect both, the subsequent alignment procedure and thealgorithm of the vector components identification. Sequences too shortto be correctly aligned to the reference gene were discarded, as well asthose exceeding the maximum size reachable with NSG technology to avoidmissing part or all of the LC sequence. Once the pipeline ends for eachpool, all integration sites were collected both in files (archived inthe TIGET network attached file storage—NAS—) and in the internaldatabase, and maintained in a storage server that keeps track of themodified copies.

Integration site identification. To identify unique integration sitesand extract the excel file with all IS in rows and each sample incolumns (IS matrix) with the closest gene annotations, we run thefollowing steps: 1. Creation of the IS matrix using the program calledcreate_matrix, enabling the collision detection inter projects. Thisprogram will produce a tab-separated file (TSV); 2. Annotation of the ISmatrix file using the program annotate_bed, that will be called asfollows for each pool using the input TSV file:awk′{print″chr″%1″\t″%2″\t″%2}′TSV_FILE|tail-n|2>TSV_FILE.bed;annotate_bed-a/opt/genome/mouse/mm9/annotation/mm9.refGene.TIGET.gtf-bTSV_FILE.bed-o TSV_FILE.annotated.bed; 3. Import both annotation andmatrix file into a new Excel worksheet, here on called XLS.

Collision detection. In order to obtain a reliable dataset of ISs fromeach transplanted mouse, we filtered data from potentialcontaminations/collisions and from false positives based on sequencecounts. An additional step of data normalization was required to combineintegration sites resulting from different experiments.

The term “collision” is used to identify the presence of identical IS inindependent samples. In our experimental setting, the integration ofvector in the very same genomic position in different cells is a verylow probability event. Thus, the detection of identical ISs inindependent samples likely derives from contamination, which may occurat different stages of wet laboratory procedures (sample purification,DNA extraction, LAM-PCRs and sequencing). Although our working pipelineis designed to minimize the occurrence of inter-samples contacts, thehigh-throughput analysis of ISs intrinsically carries a certain degreeof background contamination. Identification of the extent ofcontamination between samples is crucial also because the retrieval ofthe same IS in different samples obtained from the same mouse is used insubsequent steps to make inference on biological properties of thevector-marked hematopoietic cells (i.e. multi-lineage potential andsustained clonogenic activity). Thus, we must be able to distinguish theactual occurrence of the same IS in different samples (from the samemouse) from a contamination/collision. To address these issues, weassessed the extent of shared IS among samples derived from differenttest items and mice as a way to measure the extent of collision in ouranalyses and then design rules to discard from each mouse's data setthose IS that can be ascribed to collision and minimize the likelihoodof scoring false positive when looking for shared IS between samplesfrom the same mouse. We designed a collision detection process allowingthe validation of each integration locus. The overall result should bethat, given the set I of integration loci, in case of classification ofan integration locus i in I as collision, i is discarded from I. Weapplied collision detection process between 3 independenttransplantation groups: 1. coPKR170s: mice from assay 1 euthanized at170 days after transplant with Lin⁻ cells transduced with the coRPKexpressing LV vector (coRPK 1-3). 2. EGFP: mice from assay 2transplanted with Lin⁻ cells carrying the EGFP expressing LV vector(EGFP 1-6). 3. coPKR-TC: mice from assay 2 transplanted with Lin⁻ cellstransduced with the coPKR expressing LV vector (coRPK 1-14), whose bloodand BM was analysed at different time-points, including secondaryrecipients transplanted with a pooled BM from a sub-group of primarytransplanted mice (coRPK 11-14).

Each identical IS has different sequence reads (sequence count) amongthe different mice. Sequence counts can be used to determine whethersamples from one mouse contaminated the other mice' samples based on theabundance criterion. In our rationale, an integration found in two micewill be assigned to the mouse that shows the highest abundance, while inthe other mouse it will be considered as a contaminant. Therefore, wecould identify a threshold of differential sequence count that allowsassigning a given collision to a mouse and removing from the others. Weretrieved the threshold value from our data obtaining a value of 10,meaning that for each IS, among all TI, if an IS has got an abundancevalue (percentage sequence count ratio) 10 times lower than the highestabundance value (percentage sequence count) of the other TIs, then it isdiscarded from the current TI. We applied these rules both among the TIand the selected groups, and Excel files were used to compute thecollisions detection by applying the following rules (here detailed forTI filtering but that are extended to groups filtering as well): 1.Isolating each TI, group all samples of the same TI together by summingthe sequence counts. 2. For the three TIs obtained, for each IS, computethe percentage ratio of the IS sequence count versus the overall sum ofreads for the TI. 3. Then, applying the following rule to compute thedecision step with the threshold of 10 that allowed to assigning each ISto a reliable TI.

Once an IS was detected to remove, reads of that IS were removed fromthe group so that it will no longer assigned also to that group. Thefilter described above was applied between the mice transplanted withdifferent ex-vivo transduced cell populations (one cohort of EGFPexpressing mice from assay 2, and two cohorts of coPKR mice belonging totwo independent transplantation experiments). Moreover for the coPKR-TCgroup (assay 2), the filter method mentioned above was modified in twoways: a) To better highlight the sharing of integrations betweentime-points in the context of the clonal abundance analysis we added thefollowing rule: if one integration is shared between one or more micethen the integration will be kept for all time points even if theirsequence count is less than the 10% of the maximum sequence count amongmice; b) For lineage tracking relationships we applied a more stringentfilter by eliminating the IS with a sequence count lower than 3 and the10% sequence count filter for sharing between time-points. Meaning thatan integration shared between two time points will be kept or discardedonly if is more or less than the other respectively.

Gene ontology analysis. All gene ontology analyses were made using theGREAT online software (bejerano.stanford.edu/great/public/). The webpage allow to upload the genomic coordinates of the integrations of eachdataset and calculates the enrichment levels in the tested dataset bycorrelating positional information (based on the binomial distributionanalysis for p-value calculations) and annotated function of the genesnearest to the integration sites (based on the hypergeometricdistribution analysis for p-value calculations) [Groeschel S, et al.(2011). J Inherit Metab Dis 34: 1095-1102]. Biological processes andmolecular functions of the Gene ontology database were chosen forenrichment analysis. Only the gene classes with a false discoveryrate<0.05 for both statistical analyses were considered (FIG. 20).

Data storage. All data, both row data and results, are stored in TIGETnetwork attached file storage (NAS) in the root folder, in which allalignments from the pipeline are available, as well as the abundancematrix and plots. NAS storage is secured by authentication andauthorization policies, and was built on a reliable and scalableinfrastructure using redundancy array of disks RAID 5, and it is underbackup on our CrashPlan software registered in TIGET.

Example 1 PGK-coRPK Therapeutic Lentiviral Vector Leads to a Stable AndLong-Term Correction of the Anemic Phenotype in Genetically CorrectedPKD Mice

In vivo efficacy of the PGK-coRPK LV (FIG. 2a ) was assayed bytransduction and transplantation of lineage depleted BM cells (Lin⁻cells) from PKD mice (FIG. 2b ). FIG. 2a is a schematic representationof the self-inactivating lentiviral vectors used throughout gene therapyexperiments harboring the human PGK promoter regulating the expressionof the EGFP transgene in the control vector (upper diagram) or theexpression of a codon-optimized sequence of the PKLR gene cDNA (coRPK)in the therapeutic vector (lower diagram). The coRPK sequence showed80.4% homology with the human PKLR cDNA and 76.5% homology with themouse Pklr cDNA, with no changes in the amino acid sequence. FIG. 2b isa schematic of the gene therapy protocol performed to address thefunctionality of the developed PGK-coRPK lentiviral vector. Correctionof the PKD phenotype was studied for 4 to 9 months after transplant inPB and BM through hematological analysis and metabolic profiling.Integration analysis was performed in different tissues and time-pointsfrom all mice to address LV vector safety. At 280 dayspost-transplantation, total BM from primary transplanted mice carryingthe coRPK transgene was transplanted again into lethally-irradiatedfemale PKD mice (secondary recipients) to test the stability and safetyof the engraftment. Lethally irradiated PKD mice transplanted withdeficient cells transduced with the coRPK LV showed a significantimprovement in all tested blood erythroid parameters when compared tonon-transplanted PKD littermates or to mice transplanted with cellstransduced with an EGFP LV (FIG. 3 and Table 2).

TABLE 2 Hematological variables recorded 140 days post-transplantationin peripheral blood. Group HGB (g/dL) HTC (%) MCV MCH (pg) Healthy (n =5) 14.64 ± 0.99  36.15 ± 2.64  38.20 ± 0.86 15.44 ± 0.21 PKD (n = 9)9.70 ± 0.55 28.91 ± 1.53  51.56 ± 0.50 17.23 ± 0.31 EGFP (n = 8) 6.81 ±0.68 21.32 ± 1.51  49.13 ± 0.72 15.46 ± 0.62 coRPK n = 17) 10.67 ± 0.53*31.09 ± 1.45* 45.65 ± 0.84 15.35 ± 0.53 2nd coRPK  12.40 ± 0.67** 34.11± 1.49* 46.25 ± 0.85 16.78 ± 0.23 (n = 4)

Data represent the mean±SEM and were statistically analysed bycomparison to EGFP-expressing mice using the Kruskal-Wallisnon-parametric test. *p<0.05; **p<0.01.

RBC counts increased as soon as 40 days post-transplantation (FIG. 3a )and constitutive reticulocytosis, which is one of the most common signsof PKD, was significantly reverted in mice carrying the PGK-coRPKtransgene, reaching levels close to those observed in healthy controlsfor at least 9 months after transplant (FIG. 3b ). On the contrary, PKDanimals transplanted with EGFP LV-transduced cells showed anemia andremarkable reticulocytosis in parallel with PKD mice at all time-pointsanalyzed. Hemoglobin levels (HGB), hematocrit index (HTC), meancorpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) valueswere also corrected in mice transplanted with lentivirally correctedcells when compared to non-transplanted PKD littermates (Table 2). Thishematological correction was achieved with 63.66±4.45% of donorchimerism and transduction efficacies ranging from 60% to 90% (Table 3).

TABLE 3 Relevant molecular parameters in mice transplanted withgenetically modified cells. Donor Vector copy number Transduction %chimerism (VCN/cell) Provirus⁺ % SRY⁺ Assay Groups WBC Total BM CFU CFUsPB cells 1 EGFP (n = 2) .83 ± 0.05 .42 ± 0.03 0.42 ± 0.00 57.73 ± 12.28n.d MOI coRPK (n = 3) .76 ± 0.28 .58 ± 0.34 3.07 ± 0.76 91.08 ± 3.67 n.d 1-4 2 EGFP n = 6 .56 ± 0.50 .19 ± 1.29 3.93 ± 0.98 92.32 ± 7.68 61.82 ± 3.61 MOI coRPK (n = 14) .65 ± 0.08 .99 ± 0.13 1.89 ± 0.42 62.06± 11.73 63.66 ± 4.45 10 2^(nd) coRPK .44 ± 0.08 n.d n.d 63.15 ± 0.31^(a)62.89 ± 5.61 (n = 4)

Data represent the mean±SEM, n.d, not determined. ª estimatedtransduction percentage obtained by interpolation in the linearregression built from experiment 1 (X axis: VCN/WBC, Y axis: % provirus⁺CFUs).

Transduced cells showed on average 1.65±0.08 integrated vector copiesper cell, indicating that PGK-coRPK LV-vector provided enough human RPKtransgenic expression to revert the hemolytic anemia. Remarkably, theexpression of the coRPK transgene led to an extension of erythroid cellhalf-life when compared to non-transplanted PKD mice (FIGS. 3c,d ). Onaverage, PKD mice showed a RBC half-life of 19 days, while ingenetically corrected mice this was extended to 25 days (6 days'extension) reaching values close to wild-type RBC half-life (FIG. 3d ).Thus, RBCs of coRPK-expressing mice displayed intermediate survivalkinetics between healthy and deficient control mice (FIG. 3c ), mostlikely because full chimerism was not achieved in these animals (Table3).

Nine months after transplant hematopoietic progenitors from primaryrecipients were transplanted into secondary recipients that maintainedengraftment levels (62.89±5.61%) and VCN (1.44±0.08 copies) (Table 3).Secondary transplanted recipients showed a multi-lineage hematopoieticreconstitution up to 5 months post-transplantation (FIG. 4) and asignificant improvement in all PB erythroid parameters (FIG. 5 and Table2). FIG. 4a is a diagram of the flow cytometry strategy used to identifythe different hematopoietic lineages by labeling with CD3-PE, B220-PE,B220-PECyS, Grl-Biotin and Macl-Biotin antibodies plus SAV-PE-CyS. FIG.4b depicts representative dot-plots and percentages (FIG. 4c ) of eachlineage in PB at 140 days after transplant. Bars represent the averagepercentage±SEM of healthy (n=2, black bar) and PKD mouse (n=2, grey bar)controls and secondary transplanted mice expressing the coRPKtherapeutic transgene (n=4, scratched bar). In addition, proviralintegrations were detected in differently committed hematopoieticprogenitors (FIGS. 6a,b ) and its number remained constant over time(FIG. 6c ) demonstrating the stability of the genetic correction andhighlighting the safety of the PGK-coRPK LV. FIG. 6a shows the vectorcopy number per cell in BM CFUs from individual transplanted mice at 120to 170 days after transplant. Transduction and chimerism percentages arealso shown. FIG. 6b shows provirus copy number in cells from differenthematopoietic compartments. Columns represent the average±SEM of thedifferent groups of transplanted mice. FIG. 6c shows the kinetics ofproviral integrations in BM cells from individual transplantedEGFP-expressing mice (grey lines) and mice carrying the coRPK transgene(black lines).

Example 2 Lentiviral-Derived RPK Expression Normalizes ErythroidDifferentiation and Allows the Production of Functional MatureErythrocytes

PKD mice show a characteristic expansion of the erythroid compartmentcaused by the compensatory erythropoiesis mechanism (Min-oo et al 2004).The study of the erythroid differentiation pattern in transplanted miceindicated that the ectopic RPK expression reverted this mechanism (FIGS.7a,b ). PKD and EGFP-expressing mice showed a predominance of immatureerythroid precursors (subpopulation I: proerythroblasts andsubpopulation II: basophilic erythroblasts) in BM and spleen, and aremarkable fall in late erythroid cells (population IV: reticulocytesand mature erythrocytes), while mice transplanted with cells transducedwith the coRPK LV showed a significant reduction of immature erythroidprecursors (subpopulations I and II) in BM and spleen, and a significantincrease of the latest erythroid compartment (subpopulation IV)equivalent to healthy mice (FIGS. 7a,b ). In addition, unlike PKD andEGFP-expressing mice, those carrying the coRPK transgene showed asignificant reduction of erythropoietin (Epo) levels in plasma (FIG. 7c). FIG. 8a shows the total CFUs from spleen and FIG. 8b shows the bonemarrow at 140 days after transplant. Dots represent number of coloniesper mouse analyzed and lines represent average±SEM in each group. Datawere statistically analyzed by non-parametric Kruskal-Wallis test. Thenormalization of the erythropoiesis in PKD mice treated with thetherapeutic vector was accompanied by a reduction of the splenic numberof progenitors content to normal levels (FIG. 8a ), although no changesin the BM CFU content were noted (FIG. 8b ).

Example 3 Transplantation of Cells Transduced with the coRPK LentiviralVector Reverts Extramedullar Erythropoiesis and Organ Pathology

Due to the active destruction of RPK deficient erythrocytes, PKD andEGFP-expressing mice displayed acute splenomegaly with an increase ofspleen weight and size of over 200% when compared to healthy controls(FIGS. 9a,b ). A disorganized structure of the splenic tissue and theexpansion of the spleen red pulp were also observed in these animals,indicating an intense extramedullar erythropoiesis also supported by thepresence of erythroid cell clusters in PKD and EGFP-expressing liversections (FIG. 9c ). Remarkably, the ectopic expression of coRPKtransgene completely reverted spleen and liver pathology in geneticallycorrected mice, reducing the RBC accumulations and normalizing spleenhistological structure and size (FIG. 9). Additionally, histologicalstudies revealed the total absence of iron deposits in the liver ofgenetically corrected mice, whereas PKD mice either from thenon-transplanted group or the group transplanted with HSCs transducedwith the EGFP-carrying vector displayed an intense iron overload due tothe continuous hemolytic process (FIG. 9c ). Overall, the transplant ofgenetically corrected HSCs in PKD mice restored the normal status of theerythropoiesis and all the secondary effects caused by the hemolyticanemia.

Example 4 PGK-coRPK LV-Derived Expression Restores the GlycolysisPathway in RBCs without Modifying the WBC Metabolic Balance

Next, we performed an extensive metabolomic analysis of all transplantedand control mice to study the functional correction of RPK enzymaticactivity. Following an untargeted profiling strategy, we observedsignificant changes of glycolytic intermediates in RBCs among thedifferent groups, identifying three broad clusters of metabolitepatterns with distinct trends (FIG. 10a ). RBCs from coRPK-expressingmice showed an increase of metabolites from cluster 1 similar to healthycontrols but different from transplanted mice carrying the EGFPtransgene. Likewise, cluster 3 reflected a reduced metabolite trend ingenetically corrected mice similar to wild type mice and different fromEGFP-expressing mice. Nevertheless, cluster 2 from assay 1 showed nodifferences of metabolite profile between transplanted mouse groups(EGFP and coRPK expressing mice) (FIG. 10a ). The untargeted metabolicprofiling also showed that the genetic modification was capable ofmodifying some important glycolytic intermediates, achieving an increasein ATP (FIG. 10b ), ADP (FIG. 10c ) and pyruvate (FIG. 10d ) levels inerythrocytes isolated from mice transplanted with PGK-coRPKLV-transduced HSCs. Considering these metabolic trends, we then analysedother metabolites located closer to the PK-catalysed reaction using atargeted profiling approach. Levels of the direct PK substratephosphoenolpyruvate (PEP) (FIG. 10e ) and the 3-phosphoglycerate (3-PG)(FIG. 10f ), located upstream of the PK-catalysed reaction, approachedto that of healthy control mice. Deficient erythrocytes expressing thecoRPK transgene also produced an increase of D-lactate (FIG. 10g ), thefinal product of the anaerobic glycolysis, when compared to PKD and EGFPexpressing mice. To test whether the compensation in the glycolyticmetabolites was a result of the normalization in the PK activity inmature erythrocytes, we measured the activity of this enzyme andnormalized it in relation to the Hexokinase activity in order to avoidthe influence of high amounts of reticulocytes in the deficient animals.RBCs were purified through a cellulose column to prevent leukocyte PKactivity contamination. A complete compensation of PK activity wasobserved in the animals expressing the coRPK that reach ratios similarto those obtained from wild type healthy animals and from a normalhealthy blood donor volunteer (FIG. 11). FIG. 11a shows Pyruvate Kinaseactivity, FIG. 11b shows Hexokinase activity and FIG. 11e shows ratio ofPyruvate Kinase and Hexokinase enzymatic activities in RBCs from controlmice and mice transplanted with transduced cells. RBCs were purifiedfrom blood samples through a cellulose column to avoid leukocyte PKactivity contamination and subjected to enzyme activity evaluation.Black bars, healthy mice (n=2); white bars, mice transplanted with cellstransduced with the EGFP expressing vector (n=3); scratched bars, micetransplanted with cells transduced with the coRPK expressing vector(n=3). Checkered bars represent values from a healthy volunteer (n=1).Data represent the average±SEM of each group.

Principal component analysis showed that metabolite pattern of RBCs wasdifferent depending on the group and markedly different to WBC profile(FIG. 12a ). On the contrary, WBC sub-groups clustered together withvery little difference among groups, indicating no changes in themetabolic balance of leukocytes when expressing the ectopic coRPK (FIG.12a ). Additionally, specific metabolite changes observed in the RBCuntargeted profiling were not present in WBCs (FIGS. 12b-d ).

Example 5 PGK-coRPK LV Transduced Cells Render Polyclonal HematopoieticReconstitution without Evidence of Vector Genotoxicity

Integration profile of LVs carrying either the coRPK or the EGFPtransgene was analysed in transplanted mice. Resulting from thegenome-wide integration profile of LVs, each insertion creates a uniquegenetic mark that can be used to track the clonal behaviour inindividual transduced cells. Genomic DNA (gDNA) was obtained from WBCsand from BM cells and from primary and secondary transplanted mice, aswell as from transduced cell pools before transplant (Lin cells). LinearAmplification Mediated PCR (LAM-PCR) (FIGS. 13 and 14) was used toamplify vector/genome junctions, and to identify vector insertion sites(ISs). FIG. 13 demonstrates vector integration sites were identified byLAM-PCR amplification of 3′vector LTR-genome junctions. A MultiNAautomated system was used, generating a pattern characterized by severalbands. Vector backbone derived Tsp5091 internal control band (IC) isindicated by an arrow. FIG. 14 demonstrates vector integration siteswere identified by LAM-PCR amplification of 3′ vector LTR-genomejunctions. A MultiNA automated system was used, generating a patterncharacterized by several bands. Vector backbone derived HpyCH4IV5internal control band (IC) is indicated by an arrow.

PCR products were sequenced by MiSeq Illumina platform and the obtainedsequences were mapped onto the mouse genome by bioinformatics pipelineand filtered for collisions as described in the Methods section above(FIG. 15). FIG. 15 is the general scheme of the analysis of integrationsite mapping performed in mice transplanted with genetically modifiedhematopoietic progenitors. Bone marrow and white blood cell samples fromtransplanted mice belonging to two independent experiments (Table 3) andharvested at different time-points after transplant were analyzed asdescribed in supplementary methods following the showed pipeline.

Overall, we mapped 5,173,892 sequencing reads on the transplanted mousegenome, resulting in 2,220 unique vector integration sites. The genomicdistribution of ISs from two independent experiments matched thepreviously reported LV preference for integration within transcriptionalunits (particularly within the first 50 Kb downstream of thetranscription start site—TSS—) (FIG. 16a ), showing no skewing towardsany particular chromosome in the mouse genome (FIG. 16b ). FIG. 16ashows Integration site (IS) frequency distribution around TranscriptionStart Site (TSS) of the nearest RefSeq gene, spanning 500 Kb upstreamand downstream the TSS. Numbers on the top are the number of IS detectedfor all samples and time-points. FIG. 16b shows chromosomal distributionof LV integration sites in transplanted mice expressing the EGFPtransgene (black bars) or the coRPK therapeutic transgene (grey bars),showing no skewing towards any particular chromosome.

Safety of the PGK-coRPK LV-based gene therapy was studied by clonalabundance estimations, calculating the percentage of sequence count foreach IS (a clonal mark) with respect to the total number of sequences ofthe dataset. Dot plots and heat map representations of the relativeabundance of each IS retrieved for each mouse (FIGS. 17, 18 and 19)showed strong fluctuations in clonal composition of the different miceand of in vitro cultured Lin⁻ cells. FIG. 17 is a chart of trackedshared integrations between primary and secondary recipient micecarrying the therapeutic PGK-coRPK LV vector. Integrations detected ineither mouse in any organ and at any time are pooled. Secondaryrecipients received the pooled BM from transplanted mice coRPK11 to 14.The rest of the IS detected were detected or in the primary or in thesecondary recipients. Numbers in the boxes show the representativenessin percentage of the corresponding integration in the referred mouse. Inaddition to >5% filter applied on integration analysis, all integrationwith a sequence count<3 were eliminated. FIG. 19 presents dot plotrepresentation of clonal abundance of pooled integrations in each mousein bone marrow. The relative percentage (y-axis) for each integrationsite is relative to the total number of sequences reads obtained in eachdataset. Similarly to co-RPK transduced cells (FIG. 17), the graphindicates that the vast majority of transplanted mice show a polyclonalpattern of hematopoietic repopulation.

Also, it was possible to appreciate that for several samples, a smallnumber of integrations contributed to a large amount of sequence reads(FIGS. 17 and 18), revealing a polyclonal pattern of repopulation oftransduced HSCs. In addition, tracked shared integration between primarymice carrying the therapeutic PGK-coRPK LV and subsequently transplantedsecondary mice showed no strong sharing of integrations between thegroups, confirming the absence of clonal dominance (FIG. 18).

To determine whether hallmarks of insertional mutagenesis were presentin transplanted mice, we assessed the occurrence of Common InsertionSites (CIS) similar to currently ongoing LV-mediated clinical trials.CIS are insertional hotspots that may result from integration bias atthe time of transduction or in vivo selection of clones harbouringvector integrations that confer growth advantage. CIS were identifiedusing an algorithm based on Abel and cols and the Grubbs test foroutliers finding no CIS and thus no alarming signs of genotoxicity bythis readout. Moreover, gene ontology (GO) analysis revealed no skewingtowards gene classes involved in cancer, cell proliferation orregulation of apoptosis in any of the integration datasets sorted bytissue-distribution, time point or abundance of repopulatinghematopoietic cell clones (FIG. 20). FIG. 20 represents LV genomicintegration profile. Gene Ontology (GO) analysis was performed using theGREAT software on samples from transplanted mouse. All integrationsretrieved from this study (N=2220) showed overrepresentations of thegene functions indicated on the left part of the figure. To address ifthe most abundant integrations were enriched on specific gene classes,all integration sites with a relative sequence count>5% of the entiredataset (shown in FIG. 17) were selected, showing no GO gene classesoverrepresented.

These results suggest neutrality of vector integration and demonstratethe safety of the PGK-coRPK LV in a preclinical setting.

Example 6 Human Clinical Trial

A clinical trial is conducted to evaluate safety and preliminaryefficacy of autologous hematopoietic stem cell transplantation (HSCT)using the EU/3/14/1130 medicinal product (autologous CD34+ hematopoieticstem cells transduced with the lentiviral vector containing the RPKgene) in patients with pyruvate kinase deficiency with a history ofsevere and transfusion dependent anemia refractory to splenectomy.

The ODD EU/3/14/1130 comprises a self-inactivating lentiviral vectorexpressing the codon-optimized version of the therapeutic human PKLRgene (FIG. 21).

Self-inactivating lentiviral vectors (SIN-LV) provide a more robustexpression (Ellis 2005) and are less susceptible to transcriptionalsilencing than gamma-retroviral vectors (Pfeifer, Ikawa et al. 2002).They also show a much safer integration profile (Schroder, Shinn et al.2002) (Mitchell, Beitzel et al. 2004) (Wu, Li et al. 2003), and becauseof the 400 by deletion that they carry in the 3′ LTR sequence (Miyoshi,Blomer et al. 1998) (Zufferey, Dull et al. 1998), transgene expressionis regulated by internal promoters, increasing the safety of theLV-based genetic modification.

Vector sequence of the accepted lentiviral vector also includes severalmodifications to improve transgene expression and safety in targetcells.

One modification is the use of the human phosphoglycerate kinase (PGK)promoter, already characterized by its stable in vivo activity andimproved safety properties compared to other promoters used in genetherapy (Montini, Cesana et al. 2006, Modlich, Navarro et al. 2009,Montini, Cesana et al. 2009, Biffi, Montini et al. 2013). PGK leads to amore physiological expression of the transgene and a lowersusceptibility to transcriptional silencing (Gerolami, Uch et al. 2000,Zychlinski, Schambach et al. 2008).

Another modification is a codon-optimized version of the human PKLR cDNA(coRPK) to increase mRNA stability upon transcription. For theoptimization the GeneArt® software has been used, increasing the GCcontent and removing cryptic splice sites in order to avoidtranscriptional silencing and therefore increase transgene expression.The coRPK optimized sequence showed 80.4% homology with the human PKLRgene, with no changes in the amino acids sequence of the protein.

Another modification is a mutated post-transcriptional regulatoryelement of the woodchuck hepatitis virus (Wpre), lacking any residualopen reading frame (Schambach, Bohne et al. 2006) is also included toimprove the level of expression and stability of the therapeutic gene.The backbone, promoter and Wpre* sequences of this lentiviral vector(PGK-coRPK LV) are the same as the one corresponding to the medicinalproduct “Lentiviral vector containing the Fanconi anemia A (FANCA) genefor the therapy of Fanconi anemia Type A patients” (Ref 141/2000), aswell as vector backbone used in the currently ongoing clinical trial forthe metachromatic leukodystrophy (MLD) (Biffi, Montini et al. 2013).

Mode of Action

After harvesting the CD34⁺ progenitor cells from PKD patients, eitherfrom bone marrow (BM) or mobilized peripheral blood cells, they will betransduced ex vivo with the medicinal product and the therapeutic vectorwill be integrated in the genome of the cells. Once integrated, thetherapeutic human gene (coRPK) will be transcribed and translated withindeficient cells to produce the therapeutic RPK protein that is missingor reduced in PKD mature erythrocytes. Transduced PKD hematopoieticprogenitors will be then genetically corrected, and thus able to produceRBCs with sufficient amounts of ATP to accomplish their functions (FIG.22). These genetically corrected hematopoietic progenitors (which willconstitute the medicinal product) will be then transplanted back intothe patient, where once engrafted will generate normal erythrocytes,life-long curing the disease.

The active principle will consist in a cellular suspension of correctedhematopoietic stem cells (CD34⁺) cells with the therapeutic lentiviralvector PGK.coRPK.wpre designed as orphan drug by the European Commission(ODD EU/3/14/1130) for the treatment of pyruvate kinase deficiency.Thus, this new medicament should be included in the groups of advancedtherapy developments within the gene therapy sub-class.

The active principle will be composed by of a genetically modifiedcellular suspension of at least 2×10⁶ CD34⁺ cell/Kg of body weight withat least 0.1 copies of the therapeutic vector per cell. The cells willbe suspended in a saline buffer with 2% HSA.

The final therapeutic product will be produced according to the GMPrules, so the product requirements for its liberation and its infusionin the patient will be related with the quality of the product. Inrelation to this these specifications will be cellular viability>30%,sterility (Gram test and sterility by Pharmacopeia), absence ofmycoplasma, absence of replicative competitive lentiviral particles, anddemonstration of the potency of the therapeutic potential by thedetection of the presence of at least 0.1 vector copies per cell byquantitative PCR. Additionally, investigation and research studies ofthe content of hematopoietic progenitors and the vector copy number inthese will be performed. Three independent validations will be performedwith healthy control cells to assure the procedure reaches the abovedescribed requirements.

The final product will be finished packaged in a transfer bag sealed byheat and especially for freezing and storage until its infusion to thepatient; previously, samples will be collected for the precisecorresponding quality controls.

Mobilization

Patients will be mobilized in their respective hospitals, although thetwo first patients will be mobilized in the Hospital del Niiio Jesus,Madrid (Spain). Mobilization process will consist in the administrationof recombinant stimulating factor granulocyte colony (G-CSF, Neupogen,Amgen, Thousand Oaks, Calif., USA) at doses of 12 mg/kg twice a day forup to eight days from birth, Plerixafor 4th day 240 mg/kg/d (Mozobil®,Genzyme Europe BV, Naarden, Netherlands) to four subcutaneous doses for4 consecutive days. The hematopoietic progenitor cells from peripheralblood will be collected by leukapheresis large volumes from day 5 ofmobilization through a cell separator and according to standardprotocols in Hospital Niiio Jesus in Madrid. All the instruments andsolutions are CE marked and meet the specifications of the legislationfor medical devices.

CD34⁺ Cell Purification

Consistent with the mobilization process, the apheresis will take placein the hospital where the patient is mobilized. Apheresis will beprocessed immediately to select hematopoietic progenitors (CD34⁺ cells)through MACS “magnetic cell sorting” technology (Miltenyi Biotec,Germany) which permits separation of cells by a high magnetic fieldgradient, through a powerful permanent magnet and a separation columnwith a ferromagnetic matrix. The CliniMACS (Miltenyi Biotec, BergischGladbach, Germany) system consists on a computer (CliniMACS® plusInstrument), a specific CD34⁺ selection software, a set of sterile tubes(CliniMACS Tubing Sets), magnetic-controlled reactive sterile instrument(CliniMACS CD34 Reagent) and a sterile buffer (CliniMACS PBS/EDTABuffer). The instrument and reagents used will be CE marked and willmeet the specifications of the legislation for medical devices. Duringthis phase and in later washes nonspecific immunoglobulin (intravenousFlebogamma 5% 0.5 gr, Grifols) and human albumin (human albumin Grifols®20%, Grifols) are employed, which are later removed by washing aftercentrifugation. The CD34⁺ cells will be then quantified. Microbiologicalcontrols on the products obtained will be performed by taking standardfungal, aerobic bacteria and anaerobes samples for cultures followingspecific protocols.

CD34⁺ Transduction

Transduction of the purified CD34⁺ cells with the ODD EU/3/14/1130 willbe done under GMP conditions in a time window within 48 h since theextraction of the cells from the patient (apheresis). Ex-vivo culture ofthe cells will last less than 48 hours, and will be cultured followingestablished standards including the use of properly formulated mediaX-vivo-20 (Lonza), addition of hematopoietic growth factors (100 ng/mlhrSCF, 100 ng/ml hrFlt-3, 100 ng/ml TPO and 20 ng/mL IL-3 (all fromPrepotech), Pulmozyme and controlled 5% O₂ concentration. Transductionwill be performed with a GMP lentiviral batch of the ODD EU/3/14/1130produced by VIVEbiotech (San Sebastian, Spain). After transduction cellswill be washed in X-vivo-20 (Lonza) to be finally packaged in a transferbag suitable for cryopreservation. Specific samples will be collected todetermine if the final product meets all the specifications alreadymentioned for its final liberation. Three independent validations willbe performed to evaluate the stability of the product. All products andsolutions, including the vector, will meet the specifications of thelegislation for medical devices and clinical use. Before production, allraw materials (including consumables, biological reagents and chemicalpowders) will be inspected by the Quality Control (QC) Unit of CliniStemaccording to standard operating procedures (SOPs).

Conditioning

Patients will be conditioned according to standardized and specificprotocols considered for the trial. To consider a patient forconditioning an alternative a back-up of 2×10⁶ unmanipulated CD34⁺cells/kg will be kept frozen to be used in case the prepared product donot completely reconstitute the haematopoiesis of the treated patient.

Infusion

Previous to infusion, patient eligibility will be checked to ensure theymeet the study requirements. The day of infusion, premedication andprophylactic medication used will be recorded.

Example 7 Non-Clinical Development

Previous work demonstrated the feasibility of HSC gene therapy for PKDin mice when above 25% genetically corrected cells were transplanted.These results suggest that a significant number of donor gene-correctedHSCs (Zaucha, Yu et al. 2001) and high levels of transgene expressionare needed to achieve a therapeutic effect in PKD. We have developed anew therapeutic lentiviral vector proposed for this clinical trial,harbouring the hPGK eukaryotic promoter driving the expression of thePKLR cDNA that was designated as Orphan Drug on August 2014(EU/3/14/1130). With this vector we conducted a preclinical gene therapyprotocol for PKD in a mouse model of the disease. With lentiviraldosages based on clinical standards, ectopic RPK expression was able tonormalize the erythroid compartment, correcting the haematologicalphenotype and reverting organ pathology. Metabolomic studiesdemonstrated the functional correction of the glycolytic pathway ingenetically corrected RBCs, with no metabolic disturbances observed inleukocytes. Remarkably, WBCs analyzed in parallel showed no alterationsof the metabolic balance in leukocytes when RPK is ectopically expressedunder the activity of an ubiquitous promoter such as PGK, ruling out aleukocyte metabolic advantage as possible safety concern and reinforcesthe therapeutic potential of the EU/3/14/1130 vector.

The multi-lineage reconstitution and the absence of any leukemic eventor clonal expansion in secondary recipients after the proliferativestress induced by BM re-transplant demonstrate the long-term stabilityand safety of the PGK-coRPK LV vector-based protocol. The use of thehuman PGK eukaryotic promoter that i) likely led to a more physiologicalexpression of the RPK transgene, ii) has been proven to be a weaktransactivator and iii) is being currently used in the clinical trialfor metachromatic leukodystrophy (MLD), could also account for thesafety of the whole procedure.

To assess the long-term safety of HSC gene therapy through the analysisof vector integration sites, next generation sequencing was used topredict the risk of insertional oncogenesis in HSC. More than 5,173,892sequences reads were mapped on the mouse genome to a total of 2220unique vector IS, finding no evidence of in vivo expansion or selectionof clones harboring IS. Rather, our data show the clonal composition anddynamics of hematopoiesis after transplantation of transduced HSCs inmice, suggesting a genuine and stable genetic in vivo modification ofHSC over time. Overall, the analysis of the vector integration patternemphasizes the safety properties of the PGK-coRPK LV vector thatprovides PKD genetic correction with no evidence of genotoxicity.

Example 8 Clinical Development

So far, no clinical studies have been conducted with the medicinalproduct. This is the first time protocol assistance is requested to aRegulatory Agency. Our aim is to perform a clinical trial sponsored bythe European Commission. The ForGeTPKD Consortium, composed by differentclinicians and basic researchers in Europe, has been established tofocus in PKD research and development of new therapeutic strategies.ForGeTPKD clinical trial will be the first administration of thismedicinal product in humans. It is designed as an International,Multicenter, Phase I/II Open Label Study to evaluate the Safety andEfficacy of Transplantation of Autologous CD34+ Cells Transduced Ex Vivowith a Lentiviral vector containing the red-cell type Pyruvate Kinase(RPK) gene (EU/3/14/1130) in patients with Severe Pyruvate KinaseDeficiency.

Regulatory Status

The medicinal product has no marketing authorization at present time.The objective of the PKD Consortium is to move forward to the clinicaldevelopment of the medicinal product in order to eventually receive amarketing authorization.

The mentioned final product will be produced with a lentiviral vectorthat has received the Orphan Drug Designation related to:

Indication: Treatment of Pyruvate Kinase Deficiency

Criteria: The only curative treatment for PKD is allogeneic BMT, whichhas been used in patients with transfusion-dependent severe anemiarefractory to other measures. However, allogeneic BMT is not a widelyaccepted treatment for PKD (only one patient reported in the literature(Tanphaichitr, Suvatte et al. 2000)) as it is associated with severecomplications related to intensive pre-allo-BMT conditioning bychemotherapy or chemo-radiotherapy, as well as acute and chronicgraft-versus-host disease (GVHD). Our hypothesis is that gene therapyusing autologous hematopoietic stem cells transduced with viral vectorscontaining the wild type version of the gene, provided by the ODDEU/3/14/1130, may represent a potential curative opportunity for thesepatients, avoiding the risks of GVHD, the main cause of failure of ahematopoietic progenitors transplant.

Active substance: Autologous CD34⁺ hematopoietic stem cells, transducedwith Lentiviral vector containing the red-cell type Pyruvate Kinase(RPK) gene (ODD EU/3/14/1130), expressing the wild type version of theprotein

Finished product: Frozen bag containing at least 2×10⁶ activesubstances/kg body weight of the patient, suspended in a saline bufferwith 2% HAS

Example 9 Pharmacology

Completed studies: The medicinal product developed includes severalmodifications in its sequence that provides some advantages for the genetherapy for PKD: 1) The use of a SIN-LV vector design allowed arelatively easy and safe production of viral stocks, able to efficientlytransduce HSCs; 2) the use of a weak and eukaryotic promoter such ashPGK, which is less susceptible to silencing by methylation (Gerolami,Uch et al. 2000), leads to a more physiological expression of thetransgene, achieving therapeutic levels with a viral dosage (1.65 VCN)within the clinical standards (Matrai, Chuah et al. 2010); and 3) thecodon optimized transgene sequence and the presence of the mutated Wpresequence increase transgene mRNA stability: no reporter gene wasincluded in the therapeutic vector sequence, avoiding possibleimmunogenic problems (Morris, Conerly et al. 2004); (Stripecke, CarmenVillacres et al. 1999).

The developed hPGK-coRPK LV medicinal product efficiently reverted PKDpathology in both primary and secondary deficient mice transplanted withprogenitors transduced and corrected with the ODD EU/3/14/1130. Thecorrection was achieved with cells carrying on average 1.65 copies percell of the therapeutic transgene.

Human PGK promoter was potent enough to express clinically relevantlevels of coRPK protein, restoring the hemolytic phenotype intransplanted mice.

The genetic correction was able to: Extend RBC half-life; Normalize thehematological variables and reticulocytes levels; Revert thecompensatory erythropoiesis constitutively activated in PKD mice; Rescuethe pathology in spleen and liver, remarkably reducing the ironoverload, which is one of the life-threatening complications of PKD. Inaddition, the ectopic expression of human RPK corrected the energeticdefect in RBCs without altering the metabolic balance in WBCs,emphasizing the efficacy and safety of the medicinal product.

Example 10 Ongoing Studies

Transduction of human hematopoietic progenitors from healthy donors andPKD patients for the study of: 1) efficiency of transduction of the ODDEU/3/14/1130 in human cells; 2) definition of the optimal vector copynumber/cell to get efficient and therapeutic expression of the RPKtherapeutic protein; and 3) definition of the optimal conditions to gettherapeutic transduction levels without losing hematopoietic stem cellability.

Planned studies include set up of the conditions for large scaletransduction at the GMP facility and pre-validation and 3 validationstudies to set up the optimal conditions to reach the requiredspecifications defined for the final therapeutic product.

Toxicology

Completed studies include 1) The ectopic expression of human RPKcorrected the energetic defect in RBCs without altering the metabolicbalance in WBCs; 2) Genome integration analysis of the vector hasdemonstrated that (i) The analysis of the relative abundance of specificcell clones revealed an oligoclonal hematopoietic reconstitution forsome mice, showing no clonal dominance for any primary and secondarytransplanted mice; (ii) Common Integration Sites (CIS, dense clusters ofvector integrations in defined genomic intervals), considered a hallmarkof insertional mutagenesis did not show any sign of genotoxicity,neither an abnormal enrichment of CIS over time and detected CIS fromthe two independent gene therapy experiments performed in mice were notrepresented by high sequence counts and did not preferentially targetoncogenes; (iii) Gene ontology (GO) analysis of the genes targeted bythe lentivirus integration and study of the position of the vectorintegrations in specific regions of the genome demonstrated no skewingtowards gene classes involved in cancer, cell proliferation orregulation of apoptosis; and (iv) Overall, medicinal product integrationanalysis did not show any evidence of genotoxicity.

Planned studies include 1) Analysis of Recombinant CompetentLentiviruses (RCL) production: Human T lymphocytes from healthy donorsand from PKD patients will be transduced with the ODD EU/3/14/1130 andculture in vitro for extended periods of time. Presence of viral p24protein will be analyzed in the supernatants by ELISA to evaluate thepotential generation or RCLs and 2) Biodistribution of the medicinalproduct: Mouse hematopoietic progenitors will be tranduced with the ODDEU/3/14/1130 and transplanted into lethally irradiated recipients. Onemonth post-transplant animals will be sacrificed and different organs(gonads, liver, kidney, brain, bone marrow, spleen and peripheral blood)will be analyzed for the presence of vector DNA; and 3) Vector integromein human cells: Hematopoietic progenitors from healthy donors and fromPKD patients will be transduced with the ODD EU/3/14/1130 andtransplanted into severe immunodeficient mice to allow the engraftmentand proliferation of human hematopoietic cells. At different time points(1, 2 and 3 months post-transplant) blood and BM transplants will betaken, sorted for human cells and subjected to vector integrome analysisas already performed with mouse cells.

Example 11 Human Clinical Trial

To test clinical efficacy the ForgetPKD trial will be conducted. Theproposed clinical trial aims to evaluate safety and preliminary efficacyof autologous hematopoietic stem cell transplantation (HSCT) using theEU/3/14/1130 medicinal product (autologous CD34⁺ hematopoietic stemcells transduced with Lentiviral vector containing the red-cell typePyruvate Kinase (RPK) gene) in patients with pyruvate kinase deficiencywith a history of severe and transfusion dependent anemia refractory tosplenectomy.

The primary objective is to evaluate treatment safety andtolerability/feasibility. The following endpoints will be measured inaccordance: 1) incidence and characterization of Adverse Events (AE),including: AE related to the infusion of the transduced cells, AEderived from conditioning prior to cell infusion, and AE derived fromclonal evolution related with the transduced cells; and 2) number ofpatients with stem cell engraftment at 30 days post-transplant.

Secondary objectives are to evaluate preliminary treatment efficacy. Thefollowing endpoints will be measured in accordance: Number of patientswho become “transfusion independent” at the end of the study; Inpatients who still need transfusions after treatment, ratio between themean numbers of transfusions needed within the study period (1 year)with respect to the mean number of transfusion in the last 1.5 yearsbefore baseline evaluation; Clinically significant reduction of anemia,defined as number of patients with rise in Hemoglobin levels in 2 gr/dLfrom baseline at the end of the study; Clinically significant reductionof reticulocytosis, defined as number of patients with a reduction of50% from baseline evaluation at the end of the study; and Number ofpatients with stem cell engraftment where 1% transduced cells can bedetected at 6 and 12 month post-cell infusion and at the end of thestudy.

An exploratory objective is to evaluate treatment impact on patient'squality of life. The following end-point will be measured in accordance:Improvement in quality of life from baseline at the end of the study,using a quality of life questionnaire (SF-36 for adults or PEDSQL forchildren) and its translated validated versions in the language of theparticipant countries (Italian, Dutch and Spanish).

ForGetPKD Trial is a multicenter, international trial, which will becarried out in 3 EU member states: Spain, Italy and The Netherlands.Participating centers include Reference National Investigators andInstitutions for PKD diagnosis and treatment.

The trial will represent the first administration to humans of thedescribed product. It is designed as a non-comparative, open label,single-dose, Phase I/II study.

Global Study duration will be 2 years from the first visit of the firstpatient to the last visit-last patient. This includes 1 year ofrecruitment period and 1 year of treatment period and early (immediate)follow-up. After the end of the trial, included subjects will be askedto participate in a subsequent follow up study that will monitor safetyand efficacy for up to 5 years after transplant.

According to disease incidence and study design, we are planning toinclude 6 patients in one year. This estimate has been decidedconsidering that there are 3 potential participants already identified.

Study procedures include a Screening Period, a Treatment Period and aFollow-up Period. Details of visits on each phase and associated studyprocedures are detailed below and summarized in Table 4.

TABLE 4 Period Screening Treatment Period Period Follow up period Visit1 −1 Pre- 0 Treatment 2 + 3 + 4 + 5 + Study procedures screeningBaseline period 1 m 3 m 6 m 12 m Informed consent X Inclusion/exclusionX X Criteria Medical History X Physical Examination X X X CBCs X X X X XX Biochemistry X X X X X X Coagulation X X X X X X Conditioning regimenX Transduced cells X infusion Discharge X Bone Marrow sampling X Cellengraftment in X X X X peripheral blood Average vector copy X X X Xnumber in peripheral blood Vector integration X X X X pattern inperipheral blood Cell engraftment in X X bone marrow Average vector copyX X number in bone marrow Vector integration X X pattern in bone marrowCell extraction X Availability of viable X cells Quality of life X Xquestionnaire (SF-36 or PEDSQL) Concomitant X X X X X X X MedicationAdverse Events X X X X X X X

Screening Period

Visit-1: Pre-Screening Visit

Potential candidates will be informed of the aims and characteristics ofthe trial, and written informed consent will be taken, fulfilled andsigned by the patient (or legal representative if underage), byduplicate. To be eligible for the study, patients must fulfill allinclusion criteria and none of the exclusion criteria, which will bechecked. This includes a pre-treatment procedure to mobilize and obtainviable CD34⁻ cells, to A. Store 2×10⁶ CD34⁺ cells/kg body weight toserve as a backup in case of non-engraftment, B. transduce at least6×10⁶ CD34+ cells/kg body weight with the EU/3/14/1130 vector togenerate the medicinal product and to perform all the quality controlneeded for the liberation of the medicinal product. Only patients withenough transduced cells available after liberation of the medicinalproduct (2×10⁶ transduced CD34⁺ cells/kg body weight) will be includedin the study.

The following procedures will be also performed in this visit:

Register of relevant medical o surgery history.

Register of demographic data and clinically relevant physicalexamination findings

Register of relevant concomitant medications.

Peripheral blood testing for routine cell blood counts (CBC),Biochemistry,

Coagulation determination and serology

Echocardiogram, Lung function test and thorax X-ray

Quality of life questionnaire (SF-36 or PEDSQL)

Genetic diagnosis of PKD

To be eligible for the study, patients have to fulfill all of thefollowing inclusion criteria and none of the exclusion criteria.

Inclusion criteria are male or female patients, Age>2 year old at thetime of recruitment, willing to give signed informed consent (which willbe signed by their parents or legal representative in case of childrenunder 18 years old), previous diagnosis for PKD confirmed by genetictesting, history of severe transfusion-dependent anemia, not responsiveto splenectomy, Candidate to Autologous Hematopoietic Stem CellTransplant, >2×10⁶ transduced CD34⁺ cells/kg body weight available, andtreated and followed for at least the past 2 years in a specializedcenter that maintained detailed medical records, including transfusionhistory.

Exclusion criteria are positive for presence of human immunodeficiencyvirus type 1 or 2 (HIV 1 and HIV 2), uncorrected bleeding disorder,presence of other causes of hemolysis, any prior or current malignancyor myeloproliferative or immunodeficiency disorder, immediate familymember with a known or suspected Familial Cancer Syndrome (including butnot limited to hereditary breast and ovarian cancer syndrome, hereditarynon-polyposis colorectal cancer syndrome and familial adenomatouspolyposis), in patients with previous allogeneic transplant, presence ofresidual cells of donor origin, patients with severe complications thatafter medical evaluation are considered to suffer grade III/IV cardiac,pulmonary, hepatic or renal function abnormalities, uncontrolled seizuredisorder, diffusion capacity of carbon monoxide (DLco)<50% of predicted(corrected for hemoglobin), any other evidence of severe iron overloadthat, in the Investigator's opinion, warrants exclusion, participationin another clinical study with an investigational drug within 30 days ofScreening, availability of a HLA-identical family donor for allogeneicbone marrow transplant, pregnant or breast-feeding women, patients that,according to investigator criteria, will not be able to understand studypurposes, benefits and risks and/or to comply with study procedures, anpoor functional status, evidenced by a Kamofsky Index≤80 in adults orLansky≤80 in children.

The statistical analysis of the study will be descriptive. Qualitativeendpoints will be described by frequencies and percentages. Qualitativeendpoints include the adverse events, the number of patients with stemcell engraftment, the number of patients who become “transfusionindependent”, the number of patients who have a clinically significantreduction of anemia, the number of patients who have a clinicallysignificant reduction of reticulocytosis, the number of patients withstem cell engraftment where presence of transduced cells can bedetected, and the improvement in quality of life from baseline measuredby SF-36 or PEDSQL questionnaire. Quantitative endpoints will bedescribed by mean and standard deviation or by median and quartiles.Quantitative endpoints include the reductions of anemia andreticulocytosis from baseline, the number of transfusions needed withinthe study period with respect to the number of transfusion in the last 1year before baseline evaluation, and the vector copy number inperipheral blood and bone marrow. All endpoints will be described at theend of the study.

Previous work demonstrated the feasibility of HSC gene therapy for PKDin mice when above 25% genetically corrected cell were transplanted.These results suggest that a significant number of donor gene-correctedHSCs (Zaucha, Yu et al. 2001) and high levels of transgene expressionare needed to achieve a therapeutic effect in PKD. We have developed anew therapeutic lentiviral vector proposed for this clinical trial,harboring the hPGK eukaryotic promoter driving the expression of thePKLR cDNA that was designated as Orphan Drug on August 2014(EU/3/14/1130). With this vector we conducted a preclinical gene therapyprotocol for PKD in a mouse model of the disease. With lentiviraldosages based on clinical standards, ectopic RPK expression was able tonormalize the erythroid compartment, correcting the hematologicalphenotype and reverting organ pathology. Metabolomic studiesdemonstrated the functional correction of the glycolytic pathway ingenetically corrected RBCs, with no metabolic disturbances observed inleukocytes. Remarkably, WBCs analyzed in parallel showed no alterationsof the metabolic balance in leukocytes when RPK is ectopically expressedunder the activity of an ubiquitous promoter such as PGK, ruling out aleukocyte metabolic advantage as possible safety concern and reinforcesthe therapeutic potential of the EU/3/14/1130 vector.

The multi-lineage reconstitution and the absence of any leukemic eventor clonal expansion in secondary recipients after the proliferativestress induced by BM re-transplant demonstrate the long-term stabilityand safety of the PGK-coRPK LV vector-based protocol. The use of thehuman PGK eukaryotic promoter that likely led to a more physiologicalexpression of the RPK transgene, that has been proven to be a weaktransactivator and is being currently used in the clinical trial formetachromatic leukodystrophy (MLD) could also account for the safety ofthe whole procedure.

To assess the long-term safety of HSC gene therapy through the analysisof vector integration sites, next generation sequencing was used topredict the risk of insertional oncogenesis in HSC. More than 5,173,892sequences reads were mapped on the mouse genome to a total of 2220unique vector IS, finding no evidence of in vivo expansion or selectionof clones harboring IS. Rather, our data show the clonal composition anddynamics of hematopoiesis after transplantation of transduced HSCs inmice, suggesting a genuine and stable genetic in vivo modification ofHSC over time. Overall, the analysis of the vector integration patternemphasizes the safety properties of the PGK-coRPK LV vector thatprovides PKD genetic correction with no evidence of genotoxicity.

The invention claimed is:
 1. A method of treating or preventing PyruvateKinase Deficiency (PKD) in a subject in need thereof, comprising:contacting a hematopoietic stem cell with a recombinant lentivirusvector comprising an expression cassette, wherein the expressioncassette comprises a polynucleotide sequence encoding a pyruvate kinase,liver and red blood cell (PKLR) polypeptide operatively linked to apromoter, wherein the expression cassette comprises a woodchuckhepatitis virus post-transcriptional regulatory element (Wpre), whereinthe Wpre comprises a sequence at least 90% identical to SEQ ID NO: 1;and administering the hematopoietic stem cell to the subject.
 2. Themethod of claim 1, wherein the promoter is a phosphoglycerate kinase(PGK) promoter.
 3. The method of claim 1, wherein the Wpre comprises asequence at least 95% identical to SEQ ID NO:
 1. 4. The method of claim1, wherein the Wpre comprises a sequence 100% identical to SEQ ID NO: 1.5. The method of claim 1, wherein the expression cassette comprises oneor more enhancer sequences.
 6. The method of claim 1, wherein theexpression cassette comprises a polypurine tract (PPT).
 7. The method ofclaim 1, wherein the expression cassette comprises a polyadenylation(polyA) signal sequence.
 8. The method of claim 1, wherein theexpression cassette comprises one or more of the following sequences: i)a packing signal sequence; ii) a truncated Gag sequence; iii) a Revresponsive element (RRE); iv) a central polypurine tract (cPPT); v) acentral terminal sequence (CTS); and vi) an upstream sequence element(USE).
 9. The method of claim 1, wherein following said contacting thehematopoietic stem cell with the recombinant lentivirus vector, PKLR isexpressed at detectable levels in the hematopoietic stem cell.
 10. Themethod of claim 1, wherein following said administration, thehematopoietic stem cell is engrafted in the subject.
 11. The method ofclaim 1, wherein the hematopoietic stem cell is an autologoushematopoietic stem cell obtained from the subject.
 12. The method ofclaim 9, wherein the hematopoietic stem cell is an autologoushematopoietic stem cell obtained from the subject.
 13. The method ofclaim 1, wherein the method comprises obtaining the autologoushematopoietic stem cell from the subject.
 14. A method of treating orpreventing Pyruvate Kinase Deficiency (PKD) in a subject in needthereof, comprising: administering to the subject a hematopoietic stemcell transduced with a recombinant lentivirus vector comprising anexpression cassette, wherein the expression cassette comprises apolynucleotide sequence encoding a pyruvate kinase, liver and red bloodcell (PKLR) polypeptide operatively linked to a promoter, wherein theexpression cassette comprises a woodchuck hepatitis viruspost-transcriptional regulatory element (Wpre), wherein the Wprecomprises a sequence at least 90% identical to SEQ ID NO:
 1. 15. Themethod of claim 14, wherein the hematopoietic stem cell is an autologoushematopoietic stem cell obtained from the subject.
 16. The method ofclaim 15, wherein the method comprises transducing the autologoushematopoietic stem cell with the recombinant lentivirus vector.
 17. Themethod of claim 14, wherein the promoter is a phosphoglycerate kinase(PGK) promoter.
 18. The method of claim 14, wherein the Wpre comprises asequence at least 95% identical to SEQ ID NO:
 1. 19. The method of claim14, wherein the Wpre comprises a sequence 100% identical to SEQ ID NO:1.